Microbiologically sound and stable solutions of gamma-hydroxybutyrate salt for the treatment of narcolepsy

ABSTRACT

Disclosed are formulations of gamma-hydroxybutyrate in an aqueous medium that are resistant to microbial growth. Also disclosed are formulations of gammahydroxybutyrate that are also resistant to the conversion into GBL. Disclosed are methods to treat sleep disorders, including narcolepsy, with these stable formulations of GHB. The present invention also provides methods to treat alcohol and opiate withdrawal, reduced levels of growth hormone, increased intracranial pressure, and physical pain in a patient.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/936,496, filed Nov. 9, 2015, which is a continuation of U.S.application Ser. No. 14/250,116, filed Apr. 10, 2014, which is acontinuation of U.S. application Ser. No. 13/787,437, filed Mar. 6, 2013and issued on Feb. 10, 2015 as U.S. Pat. No. 8,952,062, which is acontinuation of U.S. application Ser. No. 13/685,561, filed Nov. 26,2012 and issued on Oct. 14, 2014 as U.S. Pat. No. 8,859,619, which is acontinuation of U.S. application Ser. No. 12/913,644, filed Oct. 27,2010, which is a continuation of U.S. application Ser. No. 11/777,877,filed Jul. 13, 2007 and issued Dec. 14, 2010 as U.S. Pat. No. 7,851,506,which is a divisional of U.S. application Ser. No. 10/841,709, filed onMay 7, 2004 and issued on Aug. 28, 2007 as U.S. Pat. No. 7,262,219,which is a divisional of U.S. application Ser. No. 10/194,021, filedJul. 11, 2002 and issued on Aug. 24, 2004 as U.S. Pat. No. 6,780,889,which is a divisional of U.S. application Ser. No. 09/470,570, filedDec. 22, 1999 and issued on Oct. 29, 2002 as U.S. Pat. No. 6,472,431,which claims priority from U.S. Provisional Patent Application Ser. No.60/113,745, filed Dec. 23, 1998. These applications are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the fields of pharmaceuticalcompositions to be used in treatments, such as, sleeping disorders, suchas, e.g., narcolepsy (particularly cataplexy), drug abuse, alcohol andopiate withdrawal, a reduced level of growth hormone, anxiety,analgesia, effects in certain neurological disorders such as Parkinson'sDisease, depression, certain endocrine disturbances and tissueprotection following hypoxia/anoxia such as in stroke or myocardialinfarction, or for an increased level of intracranial pressure or thelike. The present invention particularly relates to the field ofpharmaceutical production of microbiologically resistant and chemicallystable preparations or solutions of gamma-hydroxybutyrate (GHB), alsoknown as 4-hydroxybutyrate, and the sodium salt of GHB (sodium oxybate)and other salts such as magnesium, ammonium and calcium, e.g.

II. Description of Related Art

GHB is an endogenous compound with hypnotic properties that is found inmany human body tissues. GHB is present, for example, in the mammalianbrain and other tissues. In brain the highest GHB concentration is foundin the hypothalamus and basal ganglia and GHB is postulated to functionas a neurotransmitter (Snead and Moriey, 1981). The neuropharmacologiceffects of GHB include increases in brain acetylcholine, increases inbrain dopamine, inhibition of GABA-ketoglutarate transaminase anddepression of glucose utilization but not oxygen consumption in thebrain. GHB is converted to succinate and then metabolized via the Krebscycle. Clinical trials have shown that GHB increases delta sleep andimproves the continuity of sleep (Ladinsky et al., 1983; Anden andStock, 1973; Stock et al., 1973; Laborit, 1973; Lapierre et al., 1988;Lapierre et al., 1990; Yamda et al., 1967; Grove-White and Kelman, 1971;Scharf, 1985).

GHB has typically been administered in clinical trials as an oralsolution (Lee, 1977; Mamelak, 1977; Hoes, 1980; Scharf, 1985; Scrima,1990; Gallimberti, 1992; Series, 1992; Lammers, 1993). GHB treatmentsubstantially reduces the signs and symptoms of narcolepsy, i.e. daytimesleepiness, cataplexy, sleep paralysis and hypnagogic hallucinations. Inaddition, GHB increases total sleep time and REM sleep, and it decreasesREM latency (Mamelak et al. 1973; Yamada et al., 1967; Bedard el al.,1989), reduces sleep apnea (Series et al, 1992; Scrima el al., 1987),and improves general anesthesia (Hasenbos and Gielen, 1985).

GHB has several clinical applications other than narcolepsy and sleepdisorders. GHB has been reported to reduce alcohol craving, the numberof daily drinks consumed, and the symptoms of alcohol withdrawal inpatients (Gallimberti el al., 1989; Gallimberti el al., 1992; Gessa elal., 1992). GHB has been used to decrease the symptoms of opiatewithdrawal, including both heroin and methadone withdrawal (Gallimbertiet al., 1994; Gallimberti et al., 1993). It has analgesic effects thatmake it suitable as a pain reliever (U.S. Pat. No. 4,393,236).Intravenous administration of GHB has been reported to reduceintracranial pressure in patients (Strong, A. 1984). Also,administration of GHB was reported to increase growth hormone levels inpatients (Gerra et al, 1994; Oyama et al., 1970).

A good safety profile for GHB consumption, when used long term fortreatment of narcolepsy has been reported. Patients have been safelytreated for many years with GHB without development of tolerance(Scharf, 1985). Clinical laboratory tests carried out periodically onmany patients have not indicated organ or other toxicities (Lammers,1993; Scrima, 1990; Scharf, 1985; Mamelack, 1977; Mamelak, 1979;Gallimberti, 1989; Gallimberti, 1992; Gessa, 1992). The side effects ofGHB treatment have been minimal in incidence and degree of severity,though they include sleepwalking, enuresis, headache, nausea anddizziness (Broughton and Mamelak, 1979; Mamelak et al., 1981; Mamelak etal., 1977; Scrima et al., 1989; Scrima et al., 1990; Scharf et al.,1985).

The pharmacokinetics of GHB have been investigated in alcohol dependentpatients (Ferrara et al., 1992) and in normal healthy males (Palatini etal., 1993) after oral administration. GHB possesses a rapid onset andshort pharmacological effect (Ferrara et al., 1992; Palatine et al.,1993; Lee, C., 1977; van der Bogert; Gallimberti, 1989; Gallimberti,1992; Lettieri and Fung, 1978; Arena and Fung, 1980; Roth and Giarman,1966; Vickers, 1969; Lee, 1977). In alcohol dependent patients, GHBabsorption into and elimination from the systemic circulation were fastprocesses. Virtually no unchanged drug could be recovered in the urine.There were preliminary indications that the pharmacokinetics of GHBmight be non-linear or dose-dependent (Ferrara et al., 1992). In thehealthy volunteers study, the pharmacokinetics of three rising GHB doses(12.5, 25, and 50 mg/kg) were investigated. These findings indicate thatboth the oral absorption and elimination processes of GHB werecapacity-limited though the degree of dose dependency was moderate(Palatini et al., 1993).

Organic salts and amides of GHB have been produced to reduce thephysiological side effects of GHB (U.S. Pat. No. 5,380,937). Magnesiumand calcium salt have been produced to reduce the hygroscopic nature ofGHB or powdered forms (U.S. Pat. No. 4,393,236; British Patent No.922,029). However, problems with the storage of GHB solutions stillexist. GHB degrades into gamma-butyrolactone (GBL) and possibly otherdegradants in solution depending upon the pH and other factors. Also,the contamination by microorganisms in GHB solutions rapidly surpassacceptable limits, and preservatives can adversely affect the pH andthus, GHB's stability. As a chronically used product which requires highlevels of drug, the volume of a non-concentrated product creates costand handling issues. Thus, there is an immediate need for effectivesolutions of GHB that are stable to biological or chemical degradation.

SUMMARY OF THE INVENTION

The present invention overcomes deficiencies in the prior art byproviding compositions of GHB in an aqueous medium that are resistant tomicrobial growth. These compositions are also resistant to theuncontrolled degradation of GHB into GBL or other substances. Thecompositions of the present invention are stable compositions of GHBthat improve shelf-life, and provide a titratable formulation of GHB foreasy dose measurement. In addition, the concentrated solutions embodiedin this invention reduce shipping and storage requirements and allowpatients to carry more drugs for their convenience. The presentinvention provides methods to treat a number of conditions treatable byGHB, referred to herein as “therapeutic categories.” Therapeuticcategories for the present invention include, but are not limited to,sleeping disorders, drug abuse, alcohol and opiate withdrawal, a reducedlevel of growth hormone, anxiety, analgesia, effects in certainneurological disorders, such as Parkinson's Disease, depression, certainendocrine disturbances and tissue protection following hypoxia/anoxiasuch as in stroke or myocardial infarction, or an increased level ofintracranial pressure or other conditions treatable with GHB.

The invention first provides a pharmaceutical composition of GHBrendered chemically stable and/or resistant to microbial growth in anaqueous medium. Preferred GHB salts of the present invention includesodium, ammonium and calcium. As used herein in certain embodiments,“stable” may mean resistant to degradation of GHB into its known orunknown decomposition elements. The level of GBL that is acceptable canbe up to 0.1% of the formulation as per the ICH guidelines forshelf-life determination. As used herein in certain embodiments,“resistant to microbial growth” or “resistant to microbial challenge”means that the formulations meet the criteria set by the Food and DrugAdministration and the U.S. Pharmacopoeia for products made with aqueousbases or vehicles, which for bacteria means not less than a 1.0 logreduction from the initial count at 14 days, and no increase from the 14days count at 28 days, and for yeast and molds, no increase from theinitial calculated count at 14 and 28 days. As used herein in certainembodiments, an “aqueous medium” may mean a liquid comprising more thanabout 50% water. In certain preferred embodiments, an “aqueous medium”may be a solution, suspension, gel or emulsion of GHB, with a solutionof GHB being most preferred. Preferred gels are thixotropic gels.Compositions that are resistant to microbial growth are created bydissolving or mixing GHB in an aqueous medium to a concentration orcontent of greater than of about 150 mg/ml GHB to the maximal solubilityof GHB. The solubility of GHB is up to about 750 mg/ml at roomtemperature (20° C. to about 25° C.), however, heating the aqueousmedium during preparation up to 100° C. will increase GHB solubility toat least about 1000 mg/ml. A preferred concentration or content of GHBis about 500 mg/ml.

The amount of GHB that may be mixed or dissolved into an aqueous mediumand still be resistant to microbial growth depends upon the pH of theaqueous medium. In certain embodiments the presence of a preservativemay allow the amount of GHB contained in the compositions of the presentinvention to be increased and still maintain resistance to chemicaldegradation and/or microbial growth. In one embodiment of the presentinvention, the pH of the aqueous medium of the pharmaceuticalcomposition is about 3 to about 10.

In a preferred embodiment, the pH of said aqueous medium is about 6 toabout 7.5. The pH may be from about 3.0 to about 10.3, namely of about3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6,about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9,about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2,about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5,about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8,about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10.0, about 10.1,about 10.2, or about 10.3, and all pH values between each of the listedpH values, of the aqueous media. This will produce a GHB compositionthat is resistant to microbial growth as defined by the test describedherein. As used herein, the term “about” generally means within about10-20%.

These pH values will produce compositions resistant to microbial growthin an aqueous medium if the amount of GHB added, admixed, or dissolvedis from above about 150 mg/ml to about 450 mg/ml, namely, above about150 mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190mg/ml, about 200 mg/ml, about 210 mg/ml, about 220 mg/ml, about 230mg/ml, about 240 mg/ml, about 250 mg/ml, about 260 mg/ml, about 270mg/ml, about 280 mg/ml, about 290 mg/ml, about 300 mg/ml, about 310mg/ml, about 320 mg/ml, about 330 mg/ml, about 340 mg/ml, about 350mg/ml, about 360 mg/ml, about 370 mg/ml, about 380 mg/ml, about 390mg/ml, about 400 mg/ml, about 410 mg/ml, about 420 mg/ml, about 430mg/ml, about 440 mg/ml, to about 450 mg/ml, and all amounts of GHBbetween the values listed.

At the medium to high end of the concentration or content of GHB thatmay be dissolved or mixed in the aqueous medium, the maximal pH that maybe used is reduced at room temperature. This is shown in FIG. 1, agraphical presentation of acceptable formulation ranges. At aconcentration or content of about 450 mg/ml GHB, the pH may be of about3.9 to about 10.3. At a concentration or content of about 500 mg/ml GHB,the pH may be of about 4.75 to about 10.3. At a concentration or contentof about 600 mg/ml GHB, the pH may be of about 6.1 to about 10.3. At aconcentration or content of about 750 mg/ml GHB, the pH may be of about7.0 to about 10.3. Of course, all pH and concentration or content valuesin between each of the listed pH and concentration or content values areencompassed by the invention.

Certain embodiments may be selected as sub-ranges from these values ofGHB content and aqueous medium pH. For example, a specific embodimentmay be selected as a content of about 170 mg/ml to about 440 mg/ml GHBin an aqueous medium, at a pH range of about pH 5.5 to about pH 8.7.Another example of how a range may be selected in an embodiment would bethe selection of a content of about 155 mg/ml of GHB, which is a valuebetween the above listed values, to a content of about 350 mg/ml of GHB,and the selection of a pH range of the aqueous medium, such as a pHrange of about 8.87, which is a value between the listed pH values, to apH of about 8.93, which is another value between the listed values ofpH. A third example of ranges that may be selected for a specificembodiment would be selection of a single content or concentration ofGHB, such as about 200 mg/ml of GHB, and the selection of a pH range,such as a pH of about 3.5 to about 8.2. A fourth example of ranges thatmay be selected for a specific embodiment would be selection of acontent or concentration of GHB over a range, such as about 300 mg/ml toabout 400 mg/ml, and the selection of a single pH value for the aqueousmedium, such as a pH of about 3. Another example of a range selected foran embodiment may be the selection of a single content or concentrationof GHB, such as 400 mg/ml GHB, and a single pH value of the aqueousmedium, such as pH 7.7.

Other examples of how a range of an embodiment of GHB content orconcentration may be selected include a range of GHB content orconcentration from about 200 mg/ml to about 460 mg/ml GHB, encompassingthe ranges for GHB described herein, and a range of pH for the aqueousmedium may be from about pH 4.3 to about pH 7, encompassing ranges forGHB in an aqueous medium at room temperature described herein. Anotherexample would be the selection of a range of GHB content orconcentration from about 153 mg/ml to about 750 mg/ml, and a pH range ofabout 7 to about 9, encompassing ranges between the listed values of GHBcontent and pH described herein. An example may be the selection as aGHB concentration or content of about 170 mg/ml to about 640 mg/ml in anaqueous medium, at a pH range of about pH 6.5 to about pH 7.7. Anotherexample of how a range may be selected in an embodiment would be acontent or concentration of about 185 mg/ml of GHB, which is a valuebetween the listed values, to a content or concentration of about 750mg/ml of GHB, at a pH range of about 7.87, which is a value between thelisted pH values, to a pH of about 8.91, which is another value betweenthe listed values of pH. An additional example of ranges that may beselected for a specific embodiment would be a content or concentrationof about 200 mg/ml of GHB at a pH of about 7 to about 8.2. Anotherexample of ranges that may be selected for a specific embodiment wouldbe a content or concentration of about 750 mg/ml to about 400 mg/ml at apH of about 7. Another example of ranges that may be selected for aspecific embodiment would be a content or concentration of about 300mg/ml to about 750 mg/ml at a pH of about 8.5 to about 7. Anotherexample of ranges that may be selected for a specific embodiment wouldbe a content or concentration of about 400 mg/ml to about 600 mg/ml at apH of about 9 to about 5.8. And so forth. Thus, all ranges of pH and GHBconcentration or content that can be selected from the values herein andas would be understood by those of ordinary skill in the art, areencompassed by the present invention.

The chemical stability of GHB is affected by pH, with compositions ofGHB in an aqueous medium with a pH below about 6 being less effective inmaintaining the chemical stability of GHB. Compositions with a pH ofgreater than about 6.0 are preferred to produce chemically stableformulations of GHB. Thus, a preferred range to produce chemicallystable GHB would be from about pH 6 to about pH 9. However, allconcentrations or content of GHB in an aqueous medium, as describedherein, and as would be understood by those of ordinary skill in theart, may be selected to produce compositions of the present invention.

Additionally, the ranges described above are for a composition at roomtemperature, which is defined herein as from about 20° C. to about 25°C., namely, about 20° C. about 21° C., about 22° C., about 23° C., about24° C., to about 25° C. Within the values and ranges of pH describedabove, the ranges of concentration or content of GHB may increase attemperatures greater than room temperature. Thus, the maximal content orconcentration of GHB in an aqueous medium at a temperature of from about26° C. about 100° C., namely about 26° C., about 27° C., about 28° C.,about 29° C., about 30° C., about 31° C., about 32° C., about 33° C.,about 34° C., about 35° C., about 36° C., about 37° C., about 38° C.,about 39° C., about 40° C., about 41° C., about 42° C., about 43° C.,about 44° C., about 45° C., about 46° C., about 47° C., about 48° C.,about 49° C., about 50° C., about 51° C., about 52° C., about 53° C.,about 54° C., about 55° C., about 56° C., about 57° C., about 58° C.,about 59° C., about 60° C., about 61° C., about 62° C., about 63° C.,about 64° C., about 65° C., about 66° C., about 67° C., about 68° C.,about 69° C., about 70° C., about 71° C., about 72° C., about 73° C.,about 74° C., about 75° C., about 76° C., about 77° C., about 78° C.,about 79° C., about 80° C., about 81° C., about 82° C., about 83° C.,about 84° C., about 85° C., about 86° C., about 87° C., about 88° C.,about 89° C., about 90° C., about 91° C., about 92° C., about 93° C.,about 94° C., about 95° C., about 96° C., about 97° C., about 98° C.,about 99° C., to about 100° C. may be from about 750 to about 1 g/ml,namely to about 751 mg/ml, about 760 mg/ml, about 770 mg/ml, about 780mg/ml, about 790 mg/ml, about 800 mg/ml, about 810 mg/ml, about 820mg/ml, about 830 mg/ml, about 840 mg/ml, about 850 mg/ml, about 860mg/ml, about 870 mg/ml, about 880 mg/ml, about 890 mg/ml, about 900mg/ml, about 910 mg/ml, about 920 mg/ml, about 930 mg/ml, about 940mg/ml, about 950 mg/ml, about 960 mg/ml, about 970 mg/ml, about 980mg/ml, about 990 mg/ml, to about 1000 mg/ml. At temperatures below roomtemperature, the solubility of GHB may decrease, and compositions atlower temperature and solubility of GHB at the pH values and rangesdescribed herein are also encompassed by the invention. Additionally,differences of atmospheric pressure may also increase or decrease thesolubility of GHB within the ranges described, and embodiments of theinvention with an increased or decreased content of GHB due to changesin pressure are also encompassed by the invention. Of course, it isunderstood that the present invention encompasses embodiments of GHBconcentration or content in an aqueous medium at higher or lowertemperature within the values described herein, such as about 980 mg/mlto about 200 mg/ml at 95° C. GHB at a pH of about 9 to about 7.5. Orabout 150 mg/ml GHB at about 17° C. at about pH 6 to about pH 7. And soforth. Thus, all ranges of pH and GHB content that can be selected atvarious temperatures and pressures from the values above, and as wouldbe understood by those of ordinary skill in the art, are encompassed bythe present invention.

In certain other embodiments of the present invention, thepharmaceutical composition may comprise a pH adjusting or bufferingagent. Such agents may be acids, bases, or combinations thereof. Incertain embodiments, the acid may be an organic acid, preferably acarboxylic acid or alphahydroxy carboxylic acid. In certain otherembodiments, the acid is selected from the group including, but notlimited to, acetic, acetylsalicylic, barbital, barbituric, benzoic,benzyl penicillin, boric, caffeine, carbonic, citric, dichloroacetic,ethylenediaminetetra-acetic acid (EDTA), formic, glycerophosphoric,glycine, lactic, malic, mandelic, monochloroacetic, oxalic,phenobarbital, phenol, picric, propionic, saccharin, salicylic, sodiumdihydrogen phosphate, succinic, sulfadiazine, sulfamerazine,sulfapyridine, sulfathiazole, tartaric, trichloroacetic, and the like,or inorganic acids such as hydrochloric, nitric, phosphoric or sulfuric,and the like. In a preferred embodiment, the acid is malic orhydrochloric acid. In certain other embodiments, the pH adjusting agentmay be a base selected from the group including, but not limited to,acetanilide, ammonia, apomorphine, atropine, benzocaine, caffeine,calcium hydroxide, cocaine, codeine, ephedrine, morphine, papaverine,physostigmine, pilocarpine, potassium bicarbonate, potassium hydroxide,procaine, quinine, reserpine, sodium bicarbonate, sodium dihydrogenphosphate, sodium citrate, sodium taitrate, sodium carbonate, sodiumhydroxide, theobromine, thiourea or urea. In certain other embodiments,the pH adjusting agent may be a mixture of more than one acid and/ormore than one base. In other preferred embodiments, a weak acid and itsconjugate base are used to form a buffering agent to help stabilize thecomposition's pH.

In certain embodiments, the composition may contain one or more salts. A“salt” is understood herein to mean certain embodiments to mean acompound formed by the interaction of an acid and a base, the hydrogenatoms of the acid being replaced by the positive ion of the base.Various salts, including salts of GHB, are also encompassed by ***theinvention, particularly as pH adjusting or buffering agents.Pharmaceutically acceptable salts, include inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asmalic, acetic, oxalic, tartaric, mandelic, and the like. Salts formedcan also be derived from inorganic bases such as, for example, sodium,potassium, silicates, ammonium, calcium, or ferric hydroxides, and suchorganic bases as isopropyamine, trimethylamine, histidine, procaine andthe like. Alkali metal salts such as lithium, potassium, sodium, and thelike may be used, preferably with an acid to form a pH adjusting agent.Other salts may comprise ammonium, calcium, magnesium and the like. Inone embodiment, a salt of GHB comprising an alkali metal may be combinedwith an acid to create a composition that achieves the desired pH whenadmixed with an aqueous medium. In another embodiment, a weak base maybe combined with GHB to create a composition that achieves the desiredpH when admixed with an aqueous solution. Of course, other salts can beformed from compounds disclosed herein, or as would be known to one ofordinary skill in the art, and all such salts are encompassed by theinvention.

In certain embodiments, excipients may be added to the invention. An“excipient” as used herein shall mean certain embodiments which are moreor less inert substances added as diluents or vehicles or to give formor consistency when the remedy is in a solid form, though they may becontained in liquid form preparations, e.g. syrups, aromatic powders,honey, and various elixirs. Excipients may also enhance resistance tomicrobial growth, and thus act as a preservative. Such excipientsinclude, but are not limited to, xylitol, mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, cellulose derivatives,magnesium carbonate and the like.

In certain embodiments, the pharmaceutical composition may contain apreservative. A “preservative” is understood herein to mean certainembodiments which are substances added to inhibit chemical change ormicrobial action. Such preservatives may include, but are not limitedto, xylitol, sodium benzoate, methylparaben, propyl gallate BP, sorbicacid, chlorobutanol, dihydroacetic acid, monothioglycerol, potassiumbenzoate, propylparaben, benzoic acid, benzalkonium chloride, alcohol,benzoic acid, benzalkonium chloride, benzethonium chloride, benzylalcohol, butylparaben, cetylpyridinium chloride, ethylenediamine,ethylparaben, ethyl vanillin, glycerin, hypophosphorus acid,methylparaben, phenol, phenylethyl alcohol, phenylmercuric nitrate,propylparaben, sassafras oil, sodium benzoate, sodium propionate,thimerosal and potassium sorbate. Preferred preservatives may beselected from the group comprising, but not limited to, xylitol, sodiumbenzoate, methylparaben, propylparaben and potassium sorbate. Xylitol isparticularly preferred in certain compositions of the invention, becauseit acts as an preservative and a sweetener, is a caries preventative, isless laxative than other sweeteners, and is recommended for diabetics.

In certain embodiments, the pharmaceutical composition may also containan antioxidant. An “antioxidant” is understood herein to mean certainembodiments which are substances that inhibits oxidation. Suchantioxidants include, but are not limited to, ascorbyl palmitate,butylated hydroxyanisole, butylated hydroxytoluene, potassiummetabisulfite, sodium metabisulfite, anoxomer and maleic acid BP.

In certain embodiments, the pharmaceutical composition may also containa flavoring agent. A “flavoring agent” is understood herein to meancertain embodiments which are substances that alters the flavor of thecomposition during oral consumption. A type of “flavoring agent” wouldbe a sweetener. Preferred sweeteners or flavoring agents would bemicrobially non-metabolizable. Especially preferred sweeteners orflavoring agents would be carbohydrates such as xylitol and sorbitol.Such flavoring agents include, but are not limited to, acacia syrup,anethole, anise oil, aromatic elixir, benzaldehyde, benzaldehydeelixir-compound, caraway, caraway oil, cardamom oil, cardamom seed,cardamom spirit, cardamom tincture-compound, cherry juice, cherry syrup,cinnamon, cinnamon oil, cinnamon water, citric acid, citric acid syrup,clove oil, coca, coca syrup. coriander oil, dextrose, eriodictyon,eriodictyon fluidextract, eriodictyon syrup aromatic, ethyl acetate,ethyl, vanillin, fennel oil, ginger, ginger fluidextract, gingeroleoresin, glucose, glycerin, glycyrrhiza, glycyrrhiza elixir,glycyrrhiza extract, glycyrrhiza extract-pure, glycyrrhiza fluidextract,glycyrrhiza syrup, honey, non-alcoholic elixir, lavender oil, citrusextract or oil, lemon oil, lemon tincture, mannitol, methyl salicylate,nutmeg oil, orange-bitter-elixir, orange-bitter-oil, orange flower oil,orange flower water, orange oil, orange peel-bitter,orange-peel-sweet-tincture, orange spirit-compound, compound, orangesyrup, peppermint, peppermint oil, peppermint spirit, peppermint water,phenylethyl alcohol, raspberry juice, raspberry syrup, rosemary oil,rose oil, rose water, saccharin, saccharin calcium, saccharin sodium,sarsaparilla syrup, sorbitol solution, spearmint, spearmint oil,sucrose, syrup, thyme oil, tolu balsam, tolu balsam syrup, vanilla,vanilla tincture, vanillin or wild cherry syrup.

Salts, excipients, pH adjusting agents such as acids, bases andbuffering agents, flavoring agents, and other agents that may becombined with the compositions of the present invention, or may be usedto prepare the compositions of the present invention, are well known inthe art, (see for example, “Remington's Pharmaceutical Sciences” 8th and15th Editions, and Nema et al., 1997, incorporated herein in theirentirety), and are encompassed by the invention.

In certain other embodiments, the pharmaceutical composition comprisesGHB, a pH adjusting or buffering agent, and an aqueous medium, whereinthe components are admixed (sequentially or simultaneously) to preparesaid pharmaceutical composition. The pH adjusting or buffering agent andaqueous medium may be any described herein.

The invention also provides a method of preparing a chemically stableand microbial growth-resistant pharmaceutical composition for thetreatment of a condition responsive to GHB, comprising admixing GHB anda pH-adjusting or buffering agent in an aqueous medium. In certainembodiments, the method of preparing the pharmaceutical compositionfurther comprises admixing a preservative with the pharmaceuticalcomposition. Other components, such as flavoring agents, salts, and thelike, may be added to the composition. The pH adjusting or bufferingagent, aqueous medium, preservative, flavoring agents, salts, or otheringredient may be any described herein.

In certain other embodiments, the method of preparing the pharmaceuticalcomposition comprises admixing GHB, a pH adjusting or buffering agent,and an aqueous medium soon before administration to a patient suspectedof having a condition responsive to GHB.

The invention also provides a method of treating any therapeuticcategory of disorder responsive to GHB, comprising administering to apatient suspected of having such a condition a therapeutic amount of apharmaceutical composition comprising chemically stable GHB (e.g. 1-10gms.) in an aqueous medium resistant to microbial growth. In certainembodiments, the method of treating a condition responsive to GHBcomprises a patient taking a first dosage of from about 0.1 g to about10 g, namely about 0.1, about 0.2, about 0.3, about 0.4, about 0.5,about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8,about 1-9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1,about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4,about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7,about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0,about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3,about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about9.0 about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6,about 9.7, about 9.8, about 9.9, to about 10 grams of GHB, or as neededby the patient as would be recognized by one of skill in the art. Ofcourse, it will be understood that all values in between those listed,such as 9.45 grams, 6.32 grams, etc. may be administered, and thosevalues are encompassed well. In preferred embodiments, the first dose isadministered within an hour of sleep. In preferred embodiments, a seconddose of GHB within the values described above may be administered. Thissecond dose is administered preferably within about 2.0 to about 5.0hrs, namely about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1,about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4,about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, to about 5.0hours after the first dose, though it may be administered at a timeoutside of the preferred range.

In certain embodiments, a second pharmaceutical may be administered withthe composition of GHB. Such a second pharmaceutical may be e.g., astimulant administered within the same 24 hour period as the first doseof GHB. The stimulant may be, e.g., but not limited to, methylphenidateor pemoline to counter the residual effects of GHB treatment duringperiods of wakefulness. In certain embodiments, the method of treating asleep disorder may include the discontinuation of other secondpharmaceuticals used to control a sleep disorder. Such secondpharmaceuticals may include, but are not limited to, a tricyclicantidepressant.

In certain embodiments, the invention provides a method of treating anyappropriate therapeutic category of disorder, by administration of GHBcompositions of the present invention as described above for thetreatment of sleep disorders. When GHB is used in methods of treatingany therapeutic category of disorder, the GHB composition of the presentinvention may be mixed with the aqueous medium, and optionally pHadjusting or buffering agent or other additives, by the patient oradministrator soon before consumption. The patient may prepare thecomposition within a few minutes to hours before administration.Alternatively, one or more of the components may be premixed for readyuse. The components of the GHB composition of the present invention,GHB, an aqueous medium, pH adjusting or buffering agent, excipients,preservatives, flavoring agents, and/or other components or additivesmay be stored in a container means suitable to aid preservation.Preferably, the container means is in the form of a set. A “set” as usedherein certain embodiments is one or more components of the compositionpackaged in a container or other suitable storage means.

The present invention also provides a set for the treatment of acondition responsive to GHB comprising, in suitable storage means, GHBand a pH adjusting or buffering agent. In certain embodiments, the GHBand the pH-adjusting or buffering agent are separately packaged. Incertain other embodiments the GHB and the pH adjusting or bufferingagent may be mixed. The set may contain an aqueous medium. In certainother embodiments, at least one component selected from the groupincluding, but not limited to, GHB, the pH-adjusting or buffering agentand/or an aqueous medium is separately packaged. In certain otherembodiments, at least two of the components selected from the groupcomprising GHB, a pH adjusting or buffering agent and an aqueous mediumare mixed together. In some embodiments, the set further contains apreservative. Such a set may have one, two, or more components from thegroup comprising GHB, a pH-adjusting or buffering agent, an aqueousmedium or a preservative packaged separately. Such a set may have two ormore components mixed together. Thus, both liquid and dry formulationsof GHB and other components may be packaged in a set for mixing beforeadministration, or one or more components may be premixed and packagedtogether with other components, or all the components may be premixedand packaged in a set.

It is understood that the compositions of the present invention,including those in a set, may be dispersed in a pharmaceuticallyacceptable carrier solution as described below. Such a solution would besterile or aseptic and may include water, co-solvent vehicle buffers,isotonic agents, pharmaceutical aids or other ingredients known to thoseof skill in the art that would cause no allergic or other harmfulreaction when administered to an animal or human subject. Therefore, thepresent invention may also be described as a pharmaceutical compositionof GHB with increased stability in a pharmaceutically acceptable carriersolution.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Also as used herein, the term“a” “an” or “the” is understood to include the meaning “one or more”.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. The Range of Gamma-Hydroxybutyrate's Resistance to MicrobialGrowth and Chemical Stability in Aqueous Solution. The ordinate is thepH of solutions of GHB. The axis is the concentration (mg/ml) of GHB inaqueous solution. The region below the diagonal line [/] is the range ofGHB solubility at room temperature. Greater solubility can be achieved,up to 1 g/ml, by heating the solution up to 100° C.

FIG. 2 illustrates the concentration and volume of GHB solution that apatient administers (see also Table 4).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. Formulations ofGamma-Hydroxybutyrate

A. Microbial Growth and Gamma-Butyrolactone Formation

The present invention arises from the discovery of chemically stable andmicroorganism resistant formulations of GHB in an aqueous medium,preferably a solution, and the efficacy of these formulations in thetreatment of therapeutic categories of disorders, such as narcolepsy andother sleep disorders. Specifically, GHB is prepared at a concentrationgreater than about 150 mg/ml in an aqueous medium, up to the limits ofGHB's solubility or retention in an aqueous medium, to produce thecompositions of the present invention.

The maximum solubility of GHB is affected by the pH of the aqueousmedium. At about pH 4, the maximum amount of sodium-GHB that can bedissolved is about 450 mg/ml. The value of pH that is conducive to GHBsolubility increases, as is shown at FIG. 1, so that the minimal pH thatwill dissolve 750 mg/ml GHB was found to be about pH 6.8. This is shownin Table 1.

TABLE 1 Limits of Sodium Oxybate Solubility ID Sodium Oxybate Maximum ASolubility pH of Solution Temperature B 450 mg/cc pH 4 (HCl) 25° C 500mg/cc pH 5 (HCl) 25° D 600 mg/cc pH 6 (HCl) 25° E 750 mg/cc pH 6.8 (HCl)25° F   750 mg/cc + pH 10.3 25° G 1000 mg/cc  pH unadjusted 65° Soluble25° Gel

The pH of the aqueous medium also affects the resistance of thecomposition to microbial growth at about 500 mg/ml GHB. GHB at thisconcentration in an aqueous medium that is between about pH 5 and pH 9is resistant to microbial growth, with compositions at about pH 6 toabout pH 7.5 being particularly resistant to microbial growth. However,at concentrations of GHB greater than about 750 mg/ml above about pH7.5, the resistance to microbial growth is reduced. This is shown atTable 2.

TABLE 2 Microbial Challenge Data Summary ID Sodium Oxybate MicrobialChallenge H Concentration pH of Solution Result I 750 mg/cc 7.5 (HCl)pass J 500 mg/cc 6.0 (HCl) pass K 500 mg/cc + Excipients 6.0 (MalicAcid) pass (Xylitol) L 500 mg/cc 9.0 (HCl) pass (borderline aspergillus)M 150 mg/cc (BDL 1995) 5.0 (HCl) fail (aspergillus only) N 150 mg/cc(BDL 1995) 7.0 (HCl) fail (aspergillus & staph) O 150 mg/cc (BDL 1995)3.0 (HCl) fail (aspergillus only) P 150 mg/cc (BDL 1995) 10.3 fail(aspergillus & (unadjusted) staph) Q 500 mg/cc 6.0 (Malic Acid)discontinued R 500 mg/cc 7.5 (Malic Acid) pass S 500 mg/cc (May ′98) 9.0(Malic Acid) discontinued T 500 mg/cc (May ′98) 7.5 (HCl) pass* UOthers: 5.0-9.0 pending 200 mg/cc-800 mg/cc *pass is generally definedas: For Category 1C Products Bacteria: Not less than 1.0 log reductionfrom the initial count at 14 days, and no increase from the 14 days'count at 28 days. Yeast and Molds: No increase from the initialcalculated count at 14 and 28 days.

The data from Table 1 and Table 2 are graphically shown in FIG. 1. Theconcentration of GHB in the composition, when evaluated in relationshipto the pH, affects the resistance of the GHB composition to microbialchallenge. Compositions of GHB at or below 150 mg/ml are poorlyresistant to microbial challenge from a pH range of about pH 3 to aboutpH 9. However, concentrations of GHB of greater than about 150 mg/ml, upto about 1000 mg/ml of GHB, are believed to be suitably resistant tomicrobial contamination at these pH ranges.

The chemical stability of GHB is affected by pH. Accordingly, the methodfor preparing GHB, as described herein, particularly as disclosed in thespecific examples, varies with pH. GBL begins to form if the pH is about6 or less. Compositions with a pH of greater than about 6.0 arepreferred to produce chemically stable formulations of 15 GHB. Thus, apreferred range to produce chemically stable GHB would be from about pH6 to about pH 9. However, any pH or range of pH values where aclinically acceptable amount of GBL is produced is also contemplated asbeing preferred, and is encompassed by the present invention. The rangeof GBL could be regulatorily broadened with availability of sufficienttoxicological data.

In certain embodiments of the invention, a pH-adjusting agent may beadded to the composition. The choice of a pH adjusting agent may affectthe resistance to microbial challenge and/or the stability of GHB, asmeasured by the reduction in assayable GHB. Compositions of GHB, pHadjusted with malic acid are resistant to both microbial growth andchemical degradation of GHB, and are preferred. Other pH adjusting orbuffering agents may be selected. Agents that adjust pH that areselected on this basis will undergo a taste testing study. However, anypH adjusting agent disclosed herein or as would be known to one ofordinary skill in the art is contemplated as being useful in theinvention. Of course, any salt, flavoring agent, excipient, or otherpharmaceutically acceptable addition described herein or as would beknown to one of ordinary skill in the art is contemplated as beinguseful in the invention.

Any of the above formulations may be prepared and/or packaged as apowdered or dry form for mixing with an aqueous medium before oraladministration, or they may be prepared in an aqueous medium andpackaged. After mixing with an aqueous medium, preferably to prepare asolution, these formulations are resistant to both microbial growth andchemical conversion of GHB to GBL, thereby increasing the shelf-life oftherapeutic formulations of GHB in an aqueous medium. Theseformulations-then provide an easily titratable liquid medium formeasuring the dosage of GHB to be administered to a patient.

Additional embodiments of the composition and methods of preparation aredescribed below and in the examples.

B. Pharmaceutical Compositions

1. Pharmaceutically Acceptable Carriers

Aqueous compositions of the present invention comprise an effectiveamount of GHB dissolved or dispersed in a pharmaceutically acceptablecarrier and/or an aqueous medium. The phrases “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, or a human, as appropriate.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions isnot appropriate. Supplementary compatible active ingredients can beincorporated into the compositions. For human administration,preparations should meet sterility, pyrogenicity, general safety andpurity standards as required by the Food and Drug Administration (FDA).

The GHB may be lyophilized for more ready formulation into a desiredvehicle where appropriate. The active compounds may be formulated forparenteral administration, e.g., formulated for injection viaintravenous, intraarterial, intramuscular, sub-cutaneous, intralesional,intraperitoneal or other parenteral routes. The preparation of anaqueous composition that contains a GHB agent as an active component oringredient will be known to those of skill in the art in light of thepresent disclosure. Typically, such compositions can be prepared asinjectables, either as liquid solutions or suspensions. Solid formssuitable for using to prepare solutions or suspensions upon the additionof a liquid prior to injection can also be prepared; and thepreparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including, e.g., aqueouspropylene glycol; and sterile powders for the extemporaneous preparationof sterile injectable solutions or dispersions. In all cases the formmust be sterile and must be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi.

Solutions of the active compounds as free acid or pharmacologicallyacceptable salts can be prepared in water suitably mixed withhydroxypropylcellulose and/or a pharmaceutically acceptable surfactant.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof as well as in oils. Under ordinaryconditions of storage and use, these preparation may best contain apreservative to further prevent the growth of microorganisms.

A GHB composition of the present invention can be formulated into acomposition in a neutral or salt form. Such salts can be formed from anyof the acids and bases described herein particularly depending on theparticular GHB or GHB salt used, or as would be known to one of ordinaryskill in the art.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, or the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a substance, such as lecithin (e.g. a coating),by the maintenance of the required particle size in the case ofdispersion and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by any of the preservativesdescribed herein, or as would be known to one of ordinary skill in theart, including various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.In many cases, it will be preferable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The preparation of more, or highly, concentratedsolutions for direct injection is also contemplated, where the use ofDMSO as solvent (although DMSO may not now be a permitted human drug) isenvisioned to result in extremely rapid penetration, delivering highconcentrations of the active agents to a small area.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of fluid or injected at the proposed site ofinfusion, (see for example, “Remington's Pharmaceutical Sciences” 15thEdition, pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject.

The active GHB may be formulated within a therapeutic mixture tocomprise about 100 to about 10,000 milligrams per dose. Multiple dosescan also be administered.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g., tablets or other solids; liposomalformulations; time release capsules; and any other form currently used,including cremes, which then may be admixed with an aqueous medium fororal administration.

One may also use nasal solutions or sprays, aerosols or inhalants in thepresent invention. Nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,the aqueous nasal solutions usually are isotonic and slightly bufferedto maintain a pH of 5.5 to 6.5, though other pH ranges disclosed hereinthe specific examples, such as pH 3 to about pH 9, or pH 6 to about 7.5,are contemplated. In addition, preservatives, similar to those used inophthalmic preparations, and appropriate drug stabilizers, if required,may be included in the formulation. Various commercial nasalpreparations are known and include, for example, antibiotics andantihistamines and are used for asthma prophylaxis.

The preferred oral formulations may include such normally employedexcipients, as, for example, pharmaceutical grades of xylitol, mannitol,lactose, starch, magnesium stearate, sodium saccharin, cellulose,magnesium carbonate and the like. These compositions can take the formof solutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders to be admixed with an aqueous medium. In certaindefined embodiments, oral pharmaceutical compositions will comprise aninert diluent or assimilable edible carrier, or they may be enclosed inhard or soft shell gelatin capsule, or they may be compressed intotablets, or the GHB may be packaged separately from or in combinationwith the excipients, salts, flavorings or any other components describedherein, to be admixed with an aqueous medium for oral or injectableformulations, or they may be incorporated directly with the food (i.e. abeverage) of the diet.

For oral therapeutic administration, the active compounds may beincorporated with excipients and used in the form of tablets, buccaltablets or tabs, troches, capsules, elixirs, suspensions, syrups,wafers, and the like, to be admixed with an aqueous medium. Suchcompositions and preparations should contain at least 0.1% of activecompound. The percentage of the compositions and preparations may, ofcourse, be varied and may conveniently be between about 2 to about 75%of the weight of the unit, or preferably between 25-60%. The amount ofactive compounds in such therapeutically useful compositions is suchthat a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, natural as gum tragacanth, acacia, cornstarch, orgelatin or synthetic as polyvinyl acetate; excipients, such as dicalciumphosphate; a disintegrating agent, such as corn starch, potato starch,alginic acid and the like; a lubricant, such as magnesium stearate; anda sweetening agent, such as sucrose, lactose or saccharin may be addedor a natural or synthetic flavoring agent. When the dosage unit form isa capsule for admixing with a specific volume of an aqueous medium, itmay contain, in addition to materials of the above type, a liquidcarrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with sugar, natural orsynthetic polymers, or both. A syrup or elixir may contain the activecompounds, sucrose as a sweetening agent, a preservative, a dye and/or aflavoring.

Additionally, any excipient, salt, acid, pH-mediating, adjusting orbuffering compound or agent, flavoring, solution, solvent, dispersion,glycerol, glycol, oil, antibacterial and antifungal agents, antibioticsand antihistamines, binders, disintegrating agents, lubricants,sweetening agents, or any other additive or ingredient from thoseenumerated above or in the examples, or in any pharmaceuticallyacceptable composition or carrier described herein, or as would be knownby one of skill in the art, is contemplated for use in aqueous mediumsor solid forms of the GHB compositions of the invention. One or more ofthese compositions may be packaged with GHB or packaged separately fromGHB prior to consumption. If packaged separately, useful compositions ofGHB may be obtained by mixing GHB with the other components with anaqueous medium prior to consumption. Such components may be packaged ina set, described below.

2. Sets

Therapeutic sets of the present invention are sets comprising GHB. Suchsets will generally contain, in suitable container, a pharmaceuticallyacceptable formulation of GHB. The set may have a single container, orit may have distinct container for each component, or distinct containerfor various combinations of components.

When the components of the set are provided in one or more liquidformulations, the liquid formulation is an aqueous medium, with asterile aqueous solution being particularly preferred. The GHBcompositions may also be formulated into a syringeable composition. Inwhich case, the container means may itself be a syringe, pipette, vial,ampule or other such like apparatus, from which the formulation may beapplied to an infected area of the body, injected into an animal, oreven applied to and mixed with the other components of the set.

However, the components of the set may be provided as dried powder(s).When reagents or components are provided as a dry powder, the powder canbe reconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container means.

The container means will generally include at least one vial, test tube,flask, bottle, pouch syringe or other container means, into which theGHB formulation or components thereof are placed, preferably, suitablyallocated. The sets may also comprise a second container means forcontaining a sterile, pharmaceutically acceptable buffer or otherdiluent.

The sets of the present invention will also typically include a meansfor containing the vials in close confinement for commercial sale, suchas, e.g., injection or blow-molded plastic containers into which thedesired vials are retained.

Irrespective of the number or type of containers, the sets of theinvention may also comprise, or be packaged with, an instrument forassisting with the injection/administration or placement of the GHBcomposition within the body of an animal. Such an instrument may be adrinking cup, syringe, pipette, or any such medically approved deliveryvehicle.

II. Methods of Treatment with the GHB Compositions

Because GHB has been shown to be effective in treating narcolepsy andsleep disorders (Lee, 1977; Mamelak, 1977; Hoes, 1980; Scharf, 1985;Scrima, 1990; Gallimberti, 1992; Series, 1992; Lammers, 1993), reducingalcohol craving and alcohol withdrawal symptoms, (Gallimberti et al.,1989; Gallimberti et al., 1992; Gessa et al., 1992), reducing opiatewithdrawal symptoms (Gallimberti et al, 1994; Gallimberti et al., 1993),reducing pain (U.S. Pat. No. 4,393,236), reducing intracranial pressurein patients (Strong, A. 1984), and increasing growth hormone levels inpatients (Gerra el al, 1994; Oyama et al., 1970), the formulations ofthe present invention are also contemplated to be useful in thetreatment of any of these disorders or conditions in patients. GHB hasalso been used alone as a narcotic in patients with a terminalcarcinomatous state. GHB has been used with other analgesics,neuroleptics, or with a subliminal barbiturate dose for use as ananesthesia. GHB has been used in closed cranio-cerebral trauma and as asoporific (U.S. Pat. No. 5,380,937). The inventors contemplate the useof the GHB compositions of the present invention as a narcotic,hypnotic, or as a soporific. The inventors also contemplate the use ofthe GHB compositions of the present invention in combination withanalgesics, neuroleptics or barbiturates for use as an anesthesia. TheGHB compositions of the present invention may be prepared andadministered by any of the means described herein. particularly thosedescribed in the section “Pharmaceutical Compositions” and the examples,or by any means as would be known to those of skill in the art.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Preferred Embodiments Xyrem™ Clinical Trials

The inventors developed a liquid formulation composed of GHB, xylitol,and preservative in water (XYREM®). Subsequent instability of thepreservative in this formulation and a desire to initiate clinicaltrials in a timely manner led to a change in the formulation to a foilpouch. One clinical trial utilized a twin-pouch dosage form, with oneside (pouch 1) of the foil packet containing GHB and the other side(pouch 2) containing the flavoring agents (Xylitol, [NF]; Malic Acid,NF.

Patients were instructed to open the twin-pouch with a scissors, emptythe contents into a dosing cup, add 2 ounces of water, snap the lid onthe dosing cup, shake to dissolve, and drink the entire contents of thecup. Clinical trials conducted by the inventors have been performedusing the twin-pouch dosage form.

However, the inventors have continued development of a liquid solutionand have now overcome inherent problems with particular formulationsand/or preservatives. The inventors have converted patients currentlyenrolled in a GHB open-label trial to a liquid solution composed of GHB,malic acid, and water—that is diluted with water immediately prior tooral administration.

The need for a liquid solution dosage form is further evidenced by therange of doses being used in a subsequent GHB open-label trial. Threesizes of pouches were prepared for the GHB open-label trial: 1.5 grams,3.0 grams. and 4.5 grams. The initial dose for all patients in the GHBopen-label trial was 6 grams of GHB nightly in divided doses. Dosageadjustments were permitted in the first two weeks of the trial asindicated for intolerance or lack of efficacy. The investigator waspermitted to decrease the dose of GHB to 3 grams or 4.5 grams, orincrease the dose to 7.5 grams or 9 grams nightly. After two weeks,further dosage adjustments were made if clinically indicated.

Thirty-five patients had their dose increased, and 16 palients had theirdose decreased. Patients in the lowest dose group weredisproportionately female and weighed 15 kg less than patients in theother two groups. Current dosing levels are noted below:

TABLE 3 Dosing Levels in the GHB Open-Label Trial 1.5 3.0 4.5 6.0 7.59.0 Total gram gram gram gram gram gram Number of Patients 95 0 4 10 3912 30 PerCent of Patients 100% 0% 4% 10% 41% 13% 32%

To achieve these individualized doses, it has been necessary to providea combination of different dose strengths. This complexity would be verydifficult to achieve with a marketed product. In addition, a month'ssupply of twin-pouches is quite bulky. A liquid formulation allows forease in dosing adjustment with one dosage form. In addition“child-resistant” packaging has been developed with the liquidformulation.

A number of patients have also complained about the flavor with thetwin-pouches. As follow-up the inventors sent questionnaires toparticipants in the inventors' clinical trial, and performed tastetesting in normal volunteers. The questionnaire responses, taste testingresults, and the clinical experience in narcolepsy patients of the studyadministrator have all confirmed that unflavored solutions wereacceptable.

The concentration and volume of the GHB solution that the patientadministers will be the same irrespective of whether it is dissolvedfrom the pouch or diluted from the liquid. This is illustrated in FIG. 2and Table 4:

TABLE 4 Comparison of Liquid Solution to Twin-Pouch Twin-Pouch LiquidSolution Amount of GHB 3 grams (1 pouch) 3 grams (6 mL) InactiveComponents malic acid malic acid xylitol lemon/lime flavor orange flavorFinal Concentration 50 mg/mL 50 mg/mL* Final Volume 60 mL 60 mL * Finalconcentration outside the range of the most stable formulation. Thisformulation strength may be only stable at short periods of time such as48 hours. The twin pouch version could be solubilized at a concentrationwithin the preferred range of pH and GHB concentration for longer termstorage. Apart from the elimination of the sweetener (xylitol) andflavoring, the two formulations result in identical solutions.

Conclusions

The concentration and volume of the GHB solution that the patientadministers is the same irrespective of whether it is dissolved from thepouch or diluted from the liquid. Either method may be used to produceacceptably stable solutions of GHB.

Example 2 Preferred Embodiments Self Preserving Formulations ofGamma-Hydroxybutyrate Summary of Formulation Studies—Liquid Xyrem™ I.Maximum Solubility Range

As seen in FIG. 1 and Table 1, the solubility of GHB varies with pHlevels at room temperature (25° C.). Additional amounts of GHB can besolubilized in a gel if heat is applied, in which case a 1000 mg/mlconcentration can be achieved. The inventors to contemplate that thoughthe concentrations or contents of GHB shown in FIG. 1 and Table 1 arepreferred for use, due to the ease of preparing and consuming unheatedpreparations, higher concentrations of GHB in aqueous medium may also bemade, up to 1000 mg/ml.

II. Microbial Testing

The inventors used a three factor analysis involving pH, concentrationsof GHB and the pH adjuster used. As seen in FIG. 1, and Table 2,unacceptably low resistance to microbial challenge was seen at 150 mg/mlGHB at pH 3, 5, 7, and 9.0, using HCl as the pH adjusting agent. 150mg/ml GHB at pH 10.3 without a pH adjusting agent also provedunacceptably resistant to microbial challenge. Borderline acceptablemicrobial preservativeness was seen in a solution pH adjusted with HClat 500 mg/ml GHB at pH 9. At a concentration of 500 mg/ml at pH 6.0 or7.5, adjusted with either malic acid or HCl, and 500 mg/ml at pH 9.0adjusted with HCl, the formulation is very effective in a microbialchallenge test. The inventors contemplate that a concentration ofgreater than about 150 mg/ml of GHB, up to the maximal solubility inaqueous solution of GHB, will be suitably resistant to microbialchallenge from about pH 3 to pH 10.3. Preferably, the aqueous mediumwill contain a pH-adjusting or buffering agent.

III. Gamma-Butyrolactone Degradation Range

GBL begins to form if the pH is about 6 or less with the formulationtested thus far.

A. Liquid Formulation Development

The objective of these experiments was to develop a commercialformulation for sodium gamma hydroxybutyric acid. The initialformulation for sodium gamma hydroxybutyric acid (GHB) was intended tobe an aqueous liquid formulation containing 150 mg/ml GHB, preservativesand flavoring agents. To develop this formulation, studies wereconducted to establish the: solubility of the drug in water and as afunction of pH, type and concentrations of suitable preservatives, typeand concentrations of flavor ingredients, and stability of theformulations.

1. Solubility

The feasibility of preparing formulations containing 150 mg/mL of GHB atpH 3, 5 and 7 was established. Solutions containing 150 mg/mL GHB wereprepared. The initial pH was greater than pH 7.5 and the final pH wasadjusted to 3, 5 or 7 with hydrochloric acid. The solutions wereobserved for precipitation and assayed by HPLC for GHB content. Theresults showed that no precipitation was observed and the drugconcentration was found to be 150 mg/mL by HPLC. This information wasused as the basis for additional formulation development studies.

2. Preservatives

Preservative effectiveness studies were conducted to identify a suitablepreservative for the GHB liquid formulation. The following formulationsshown in Table 5 were prepared and tested using Staphylococcus aureus(ATCC #6538), Pseudomonas aeruginosa (ATCC #9027) and Aspergillus niger(ATCC #16404).

TABLE 5 Liquid Formulations Used in Preservative Effectiveness TestingFormula- Sodium Methylparaben Potassium tion pH Benzoate PropylparabenSorbate Control 1 3 X 2 5 X 3 7 X 4 3 X 5 5 X 6 7 X 7 3 X 8 5 X 9 7 X 103 X 11 5 X 12 7 X 13 no pH X adjustment

The preservative used in each formulation is marked with an X. Theresults showed that formulations #3, 4, 6 and 9 reduced all threechallenge microorganisms by >99.99% in 48 h of contact time.Formulations #1, 5 and 7 reduced all three challenge microorganismby >99.99% in 7 days of contact time. Formulations #2, 8, 10, 11, 12 and13 did not reduce Aspergillus niger mold to >99.99%, although somereduction occurred in 7 days of contact time. Controls #10, 11, 12 and13 demonstrated activity against Pseudomonas aeruginosa.

3. Stability

Based on the results of the preservative effectiveness testing, fiveformulations were selected for stability testing. Table 6 shows thecomposition of the formulations.

TABLE 6 Liquid Formulations Used in Informal Stability Program Chemical1 2 3 4 5 Potassium 0.4 gm 0.4 gm Sorbate Sodium 1.0 gm Benzoate Methyl-0.36 gm 0.36 gm paraben Propyl- 0.04 gm 0.04 gm paraben GHB 30 gm 30 gm30 gm 30 gm 30 gm Xylitol 40 gm 40 gm 40 gm 40 gm 40 gm Water q.s. 200mL 200 mL 200 mL 200 mL 200 mL Initial pH 8.68 8.68 9.30 7.75 7.75Formulation 3.01 5.00 3.00 2.98 4.98 Adjusted pH

The formulations were packaged in 125 mL, amber PET bottles with safetylined child-resistant caps and stored upright and inverted at 60° C.,40° C./75% relative humidity (RH) and 25° C./60% relative humidity.Samples were removed from the stability chambers after 1, 2 and 3 monthsand assayed by high performance liquid chromatography (HPLC) for GHBcontent. Appearance and pH were also monitored.

Table 7 shows the results for the 3 month time point. Samples stored at60° C. changed color but samples at all other conditions remainedunchanged in color.

The pH of all formulations migrated upward over the three monthstability period at 60° C. The percent increase in pH from initial to 3months, was greater for the formulations which were initially adjustedto lower values.

For example, the migration of pH in formulations 1, 3 and 4 (adjusteddown to pH 3) were 21-30 percent across all conditions in three months.The migration of pH in formulations 2 and 5 (adjusted down to pH 5) were4.2-12 percent across all conditions in 3 months. Maintenance of pHbecomes important for long term storage since preservatives are known todegrade in formulations having pH levels above approximately pH 6.

Additionally, development of flavor systems to mask the negative tasteof preservatives is difficult.

TABLE 7 Results of Liquid Formulation Informal Stability Study at ThreeMonths Formulation # 25° C./60% RH 25° C./60% RH 40° C./75% RH 40°C./75% RH 60° C. (See Table 6) Attribute Upright Inverted UprightInverted Upright 1 % t = 0 100.7 101.6 101.2 NA NA Potassium pH 3.633.64 3.84 3.82 3.91 Sorbate (pH 3) at Appearance clear, colorless clear,colorless clear, colorless clear, colorless clear, light 3 monthsstorage yellow 2 % t = 0* 102.1 105.0 104.0 102.0 99.6 Potassium pH 5.215.28 5.55 5.56 5.61 Sorbate (pH 5) Appearance clear, colorless clear,colorless clear, colorless clear, colorless clear, light brown 3 % t = 0102.4 104.1 99.1 102.6 97.0 Sodium pH 3.60 3.74 3.78 3.75 3.79 Benzoate(pH 3) Appearance clear, colorless clear, colorless clear, colorlessclear, colorless clear, colorless 4 % t = 0 101.5 102.7 100.6 101.2 93.74 Methyl & pH 3.63 3.71 3.81 3.80 3.83 Propyl Parabens Appearance clear,colorless clear, colorless clear, colorless clear, colorless clear,colorless (pH 3) 5 % t = 0 103.1 105.8 101.9 103.1 95.6 4 methyl & pH5.22 5.55 5.55 5.56 5.60 Propyl Parabens Appearance clear, colorlessclear, colorless clear, colorless clear, colorless clear, light (pH 5)yellow *% GHB at t = 0 percent of label claim **initial time (t = 0)

4. Liquid Formulation Organoleptic Testing

Based on the above stability data and preservative effectivenesstesting, a pH 5 formulation containing potassium sorbate was selected asthe primary base formulation for flavor system development andorganoleptic testing. A pH 3 formulation containing potassium sarbatewas selected as the back-up formulation.

B. Dry Powder Formulation Development

Developing a flavor system for the primary and back-up liquidformulations proved to be difficult and a decision was made to develop adry powder formulation for reconstitution with water before consumption.This approach removed the need for a preservative system, therequirement to adjust pH to levels below pH 6, and allowed thedevelopment of a suitable flavor system.

1. Dry Powder Formulation Organoleptic Testing

To develop a flavor system for the powder formulation, severalparameters were evaluated. The flavor attributes of a GHB solution wascharacterized by a professional sensory panel. A mimic base containingsimilar sensory properties as a GHB solution for flavor system wasdeveloped. Generally Recognized As Safe (GRAS) excipients for flavorsystem development were selected. Different excipients (flavorings,sweeteners, acidulants and flow agents) in the mimic base were screened.Three flavor systems for the focus group test were selected. A preferredflavor system was optimized based on comments obtained from the focusgroup testing. This final formulation with GHB was optimized.

Based on the above activities, the following formulations in Table 8were selected for stability studies:

TABLE 8 Composition of Prototype Dry Powder Formulation CompositionIngredient (grams) Purpose GHB 3 Active Xylitol 5.5 non-cariogenicsweetener Malic acid 0.2 Acidulant Flavor 1 0.2 Flavor ingredient Flavor2 0.04 Flavor ingredient Silicon Dioxide (Cab-O-Sil ®) 0.03 Flowenhancer

2. Dry Powder Formulation Stability

A study was initiated to evaluate the stability of the above prototypeformulation in two types of foil packages (high and moderate moistureresistant) as well as the stability of GHB alone in one type of foilpackage (high moisture resistant). Table 9 shows the Lots that wereplaced on stability. The foil packages were a high moisture resistantpouch and a moderate moisture resistant pouch. The study protocol, Table10, required the samples to be stored at 40±2° C./75±5% relativehumidity for six months, and 25±2° C./60±5% relative humidity for 12months. Table 11 shows the tests, methods, number of packets/test andspecifications for the study.

TABLE 9 Dry Powder Informal Stability Study Package CompositionManufacture Package Lot Number Date Configuration Special Comments SPO#8018 A Oct. 6, 1995 Foil Packet Moderate moisture resistant pouch. SPO#8018 B Oct. 6, 1995 Foil Packet Highest moisture protection pouch. SPO#8018 C Oct. 6, 1995 Foil Packet Drug substance only. Highest moistureprotection pouch.

TABLE 10 Dry Powder Informal Stability Study Protocol Stability Time inMonths Storage Conditions 0 1 2 3 6 9 12 40 ± 2° C./75% ± 5% RH X X X X25 ± 2° C./60% ± 5% RH X X C C R R R X = Samples to be tested C =Contingency Samples R = Reduced testing; assay and H₂O only RH =Relative Humidity

TABLE 11 Dry Powder Informal Stability Tests and Specifications TestMethod Packets/Test Specification Limits Appearance Visual Use HPLCWhite to off-white free Dry Material flowing powder Appearance VisualUse HPLC Cloudy, off-white solution Reconstituted with visibleparticulates Material Rate of Visual Use HPLC Material should dissolveDissolution completely in five min with mixing Odor Olfactory Use HPLCCharacteristic Lemon/Lime odor Assay: GHB HPLC 3 90.0%-110.0% Assay:Malic HPLC Use HPLC 90.0%-110.0% Acid Impurities/ HPLC Use HPLC Not morethan 1% for any Degradants individual impurity/degra- dant and Not morethan 3% total impurity/degradants Vacuum Leak Visual 3 No Appearance ofLeaking test pH USP <791> Use HPLC For Information Moisture Karl Fisher3 Report Value - to be determined

After two months at 40±2° C./75±5% relative humidity, the potency (%label claim) of Lots SPO SO ISA and SPO 80 188 was less than 94.0%, thelower limit of the specification, whereas Lot SPO 8018C showed no lossin potency. Lots 8018A and 8018B showed approximately 96% potenciesafter 2 months at 25° C.±2° C./65%±5% relative humidity. Lot SPO 8018Cagain showed no loss in potency at this lower storage condition.

3. Appearance

After 2 months at 40±2° C./75±5% relative humidity, Lots SPO 8018A andSPO 8018B showed significant melting, whereas Lot 8018C showed nomelting. Lots SPO 8018A and SPO 8018B also showed partial melting after2 months at 25° C.±2° C./65%±5% relative humidity. Lot SPO 8018C againshowed no evidence of melting at this lower storage condition.

Based on the physical changes in state observed during the stabilitystudies, it was apparent that a solid state interaction between GHB andthe excipient blend had occurred. Since xylitol made up the majority ofthe excipient blend, it was assumed that xylitol was the primary sourceof the drug-excipient interaction. An alternative hypothesis was alsoproposed, based on the possibility that the package was mediating theinteraction between GHB and xylitol. Three studies were initiated totest these hypotheses.

4. Stability of GHB Solids in a Set Container-System

In the first study, the samples that were stored at 25±2° C./60±5%relative humidity were transferred to glass vials and then stored at40±2° C.17±5% relative humidity. In the second study, mixtures of GHBand xylitol were gently rubbed between sheets of different types of foilpackaging. The mixtures were observed for changes in physicalappearance. In the third study, different mixtures of GHB and xylitolwere prepared. Differential Scanning Calorimetry (DSC) thermograms werethen done to look for changes in the thermograms. The results of thesestudies are summarized below.

Transfer to Glass: Samples of Lot 8018A and Lot 8018C that werepreviously stored at 25±2° C./60±5% relative humidity were transferredto amber screw cap vials and stored at 40±2° C./75±5% relative humidity.Analyses similar to those shown in Table 6 were done. After 1 month, thepotency of Lot 8018A was 94.6% whereas the potency of Lot 8018C (GHBonly) was 100%. In addition, Lot 8018A also showed evidence of melting.The results supported the hypothesis that GHB and xylitol wereinteracting in the solid state and the interaction appeared to beindependent of packaging.

Foil Study: Mixtures of GHB and xylitol were placed between foldedsheets of several different foil packaging materials. Slight adhesion ofthe mixed granules with the foil lining was observed for all of thefoils examined. No direct evidence of melting was observed, however,even when excessive force was applied to the outer foil surfaces. Thisdata suggests that the packaging material was not responsible for thesolid state interaction observed during the stability studies.

DSC thermographs were obtained for samples of GHB/xylitol containingGHB:xylitol mixtures of 33:66, 45:55 and 55 percent 45 respectively. Thescans were conducted at a scan rate of 10° C./min. The thermogramsshowed that the sample containing GHB:xylitol 33:66 showed a broadendothermic transition starting at 35° C.-40° C. Samples with higherratios of GHB:xylitol also showed broad endothermic transitions thatstarted at temperatures of 45° C.-50° C. The changes seen in thethermograms supported the hypothesis that a solid state interaction maybe occurring between GHB and. xylitol that resulted in low potencies forformulations containing mixtures of these two agents.

As a result of the changes seen in the DSC thermograms for differentmixtures of GHB:xylitol, a study was initiated to investigate thestability of a formulation containing GHB:xylitol excipient blend 55:45.A formulation containing GHB:xylitol excipient blend 33:66 was used as acontrol sample. The formulations were packaged in glass vials and storedat 50° C., 40±2° C./75±5% relative humidity and 25±2° C./60±5% relativehumidity. The appearance and potency of the formulations were monitoredthrough analyses of stability samples. The stability study also showedpotency losses after 1 month at 40° C.±2° C./75±5% relative humiditywith both the 50/50 GHB:xylitol ratio as well as the original 33/66ratio formulation. Partial evidence of melting was also observed in bothformulations.

Studies with mixtures of GHB:xylitol excipient blend indicated that themixture was incompatible in the solid state. However, when prepared asan aqueous solution, these mixtures were chemically compatible. Usingthis information, a decision was made to package the GHB formulation indual pouches; one pouch containing GHB alone and the other containing amixture of xylitol and the other flavor ingredients. The formulation wiHcontain equal amounts of GHB and the excipient blend. This product willbe prepared, packaged, and may be checked for stability.

Example 3 The Pharmacokinetics of Gamma-Hydroxybutyrate I. StudyObjectives

The objective of this study was to assess the pharmacokinetics of GHBafter oral administration of two consecutive single doses of GHB (3g/dose; patients generally ingested the first dose of this medicationprior to bedtime and the second dose from 2.5 to 4.0 h later) tonarcoleptic patients who are maintained on a chronic regimen of GHB.

II. Study Design

This pharmacokinetic study was conducted as an open-label, single-centerinvestigation in 6 narcoleptic patients. The study design is summarizedas follows:

TABLE 12 Screening/ Treatment/Blood Washout 

Sampling 

Follow-up (1 or more days to (Two 3 g GHB oral doses, (Within 48 h afterdosing; washout, at 4 h apart; last blood sample) least 8 h) 21 bloodsamples)

Narcoleptic patients, 18 years of age or older, who volunteered for thisstudy were screened at least one day prior to the treatment phase. Eachpatient was determined to be in stable health and evaluated for thepresence of narcolepsy, defined for the purposes of this example as oneor more years of medical history of narcolepsy as evidenced by a recentnocturnal polysomnogram (PSG) and a valid score from a Muhiple SleepLatency Test (MSLT).

Patients maintained on GHB were allowed to participate. These patientshad been weaned from antidepressants, hypnotics, sedatives,antihistamines, clonidine, and anticonvulsants though a stable regimenof methylphenidate (immediate release or sustained release) was allowed.Each patient passed a pre-study physical examination (which includedhematology, blood chemistry, urinalysis, and vital signs measurements)prior to the commencement of the treatment phase.

Before oral administration of the first GHB dose, an indwelling catheterwas placed in an arm vein and a baseline blood sample was collected.Each patient then ingested a 3 g dose of GHB before bedtime. Another 3 gGHB dose was administered 4 h after the first dose. Twenty-onesequential blood samples were collected over 12 h (starting at 10 minafter the first dose and ending at 8 h after the second dose). Uponcompletion of the treatment phase, a follow-up physical examinationwhich included the measurement of vital signs was performed on eachpatient within 48 h after the last blood sample. A detailed descriptionof the trial methodology is presented in Section IV.

III. Inclusion Criteria

Patients were included in the study if they: had signed an informedconsent prior to beginning protocol required procedures; had notparticipated in such a study at an earlier date; were willing and ableto complete the entire study as described in the protocol; were 18 yearsof age or older at study entry; had not taken any investigationaltherapy other than GHB within the 30-day period prior to screening forthis study; had an established diagnosis of narcolepsy for at least oneyear with documentation from a qualified laboratory by a nocturnalpolysomnogram (PSG) and a Multiple Sleep Latency Test (MSLT) whichdemonstrated mean sleep latency to be less than 5 min and REM onset inat least 2 of 5 naps; had not been diagnosed with uncontrolled sleepapnea syndrome, defined as a sleep Apnea Index of 5 or an Apnea HypopneaIndex (AHI) greater than 10 per hour or any other cause of daytimesleepiness; and were free of any medication for their narcolepsy(including hypnotics, sedatives, antidepressants, antihistamines,clonidine, and anticonvulsants) other than GHB and methylphenidate (IRor SR). Patients admitted to this study if they were not experiencingunstable cardiovascular, endocrine, gastrointestinal, hematologic,hepatic, immunologic, metabolic, neurological, pulmonary, and/or renaldisease which would place them at risk during the study or compromisethe protocol objective; did not have neurological or psychiatricdisorders (including transient ischemic attacks, epilepsy, or multiplesclerosis) which, in the investigator's opinion, would preclude thepatients' participation and completion of this study; did not have acurrent or recent (within one year) history of alcohol or drug abuse;did not have a serum creatinine greater than 2.0 mg/dL, abnormal liverfunction tests (SGOT or SGPT more than twice the upper limit of normalor serum bilirubin more than 1.5 times normal). Female patients wereentered into the study if they were either post-menopausal (i.e. nomenstrual period for a minimum of 6 months), surgically sterilized orprovided evidence of effective birth control. Females of childbearingpotential must agree to continue to use an IUD, diaphragm, or take theiroral contraceptives for the duration of the study. Female patients ofchildbearing potential must have a negative pregnancy lest upon entryinto the study.

IV. Trial Methodology

A time and events schedule is presented in Table 12.

A. Screening Period/Washout

Six narcoleptic patients who were chronically being treated with GHBwere recruited to participate in this pharmacokinetic study. Thescreening period was at least one day prior to the treatment phase.During the screening period each patient completed the followingprocedures for the assessment of their physical condition: medicalhistory evaluation; physical examination evaluation; clinical laboratoryevaluation; inclusion criteria review. Each patient's GHB andmethylphenidate regimen also were recorded on an appropriate case reportform (CRF). The investigator also ensured that there was at least an8-hour washout period for GHB prior to the treatment.

B. Treatment Period/Blood Samples Collection

All patients were hospitalized from approximately four hours prior tofirst GHB dosing (around 6 p.m.) until the end of the treatment period(around 10 a.m. the next morning). Patients ate their dinner at theclinical research unit soon after arrival and fasted until breakfastnext morning. At least three hours elapsed between the completion ofdinner and the administration of the first GHB dose. An indwellingcatheter was placed in an arm vein of each patient for blood sampling atapproximately 30 min and 1 h before the first GHB dose and a baselineblood sample (5 mL) was collected.

The first GHB dose (3 g) was administered at around 10 p.m. Dosing ofindividual patients were staggered. The second GHB dose was administeredat 4 h after the first GHB dose (i.e. immediately after the 4 h bloodsample). The exact dosing times in each patient were recorded onappropriate CRF pages. Blood samples (5 mL each) were collected throughthe indwelling catheter into heparinized tubes at 0.2, 0.4, 0.6, 0.8, 1,1.5, 2, 3, 4, 4.2, 4.4, 4.6, 4, 8, 5, 5.5, 5, 7, 8, 10, and 12 h afterthe first GHB dose. Blood samples were processed according to theprocedures described herein. Patients were monitored for adverseexperiences throughout the study according to the specific procedures.

C. Follow-Up

Follow-up occurred within 48 h after the last blood sample had beencollected. An abbreviated physical examination which included vitalsigns measurement was performed. Adverse experiences and concomitantmedication use, if any, were assessed. Any ongoing adverse experiencesand clinically important findings in a patient were followed to theinvestigator's and/or sponsor's satisfaction before the patient wasdischarged from the study.

D. Methods of Assessment

1. Medical History

The medical history was recorded during the screening period. Thehistory included gender, age, race, height, prior reaction to drugs, useof alcohol and tobacco, history and treatment, if any, of cardiovascularpulmonary, gastrointestinal, hepatic, renal, immunologic, neurological,or psychiatric diseases and confirmation of inclusion criteria.

2. Physical Examination

Physical Examination included body system review as well as measurementof body weight and vital signs and a neurological examination.

3. Vital Signs

Vital signs measurements included recording of blood pressure, heartrate, respiration, and body temperature.

4. Clinical Laboratory

All clinical laboratory tests were performed at a local laboratory. Thelaboratory tests and analysis were required of each patient included:hematology, including hemoglobin, hematocrit, red blood cell count,white blood cell count and differential; fasting blood chemistriesincluded blood urea nitrogen (BUN), uric acid, glucose, creatinine,calcium, phosphorus, total protein, albumin, sodium. potassium, SCOT(AST), SGPT (ALT), alkaline phosphatase, lactate dehydrogenase (LDH),and total bilirubin; midstream catch urinalysis included specificgravity, pH, protein, occult blood, ketones and glucose by dipstickdetermination as well as a microscopic examination of urine sediment forRBC, WBC, epithelial cells or casts or crystals, and a urine pregnancytest. if applicable. Any laboratory parameter that was out of range andconsidered clinically significant excluded the patient fromparticipation in this study. The investigator would provide anexplanation of all observations that were significantly outside thereference range.

5. Concomitant Medication

The continued use of a fixed dose of methylphenidate immediate releaseor sustained release (IR or SR) is acceptable. The methylphenidateregimen was recorded on the appropriate case report form.

6. Adverse Experiences

An adverse experience are any undesirable event experienced by a patientor volunteer whether or not considered drug-related by the investigator.An undesirable event can be, but is not limited to, subjective symptomsexperienced by a patient or, objective findings such as significantclinical laboratory abnormalities. Adverse experience is consideredsynonymous with the term “adverse event”.

The investigators report in detail all adverse experiences and symptomsthat occurred during or following the course of trial drugadministration for up to 2 days. Included in the description was thenature of the sign or symptom; the date of onset; date or resolution(duration); the severity; the relationship to trial treatment or othertherapy; the action taken, if any; and the outcome.

A serious adverse experience is defined as one that is fatal, lifethreatening, permanently disabling, or which results in or prolongshospitalization. In addition, overdose, congenital anomaly andoccurrences of malignancy are always considered to be serious adverseexperiences. An unexpected adverse experience is one not previouslyreported.

Any serious or unexpected adverse experience (including death) due toany cause which occurs during the course of this investigation, whetheror not it is related to the investigational drug, was reported within 24h by telephone or facsimile. Appropriate authorities were to be informedif the serious or unexpected adverse experience, in the opinion ofinventors, was likely to affect the safety of other patients orvolunteers or the conduct of the trial.

7. Clinical Supplies-Study Medication

Formulation: Unit 3 g GHB doses (Lot PKI) were obtained from OrphanMedical. Each unit dose comprised twin foil pouches: one pouchcontaining GHB and the other containing a flavor excipient blend. (Table8 formulation)

Labeling: The clinical supplies for individual patients were packaged inseparate containers. Each container included two unit doses, i.e. twotwin-pouches. Clinical supplies for eight patients (including those fortwo replacement patients) were delivered to the investigator. Foiltwin-pouches were identified with a two-part label.

Dose Administration: The investigator or designee prepared the oralsolution for dosing within 30 min prior to the first oral administrationto individual patients. The contents of one twin-pouch was emptied intoa dosing cup to which, two ounces of water were added. After replacingthe lid of the dosing cup, it was gently shaken to dissolve the GHB andexcipient in water. The GHB solution was ingested in its entirety.Likewise, the second GHB dosing solution was prepared in the same mannerand was ingesting in its entirety at 4 h after the first GHB dose.

Investigational Drug Accountability: At the conclusion of the study, allclinical supplies were accounted for on the drug accountability form andunused drug supplies were returned for proper disposition.

8. Determination of Plasma GHB Concentrations

Plasma samples were analyzed for GHB by the Department of BioanalyticalChemistry (Covance (previously known as Hazelton Coming), Madison, Wis.)A gas chromatographic method with mass selective detection (GC-MSD) wasused in the analysis.

9. Data Management and Analysis

Data Base: An EXCEL data base (spreadsheet) was constructed from datarecorded on Case Report Forms (CRF) and plasma GHB concentration datasets received from Covance (Corning Hazleton). Each entry in the EXCELspreadsheet was checked against the CRFs and any data entry error foundwas corrected.

Pharmacokinetic Analysis: Pharmacokinetic parameters were determined forindividual sets of plasma GHB concentration vs. time data using thenon-compartmental routine in WinNonlin Version 1.1. The peak GHBconcentrations (C_(max)) and the times of their respectively occurrences(t_(max)) were observed values. Terminal half-life (T_(1/2)) wasobtained by log-linear regression analysis of the terminal phase ofconcentration vs. time curves. The area under the curve (AUC_(inf)) andthe area under the first moment curve (AUM_(inf)) were calculated by thelinear trapezoidal rule up to the last determined concentration andincluded extrapolated areas to time infinity. Apparent oral clearance(CL/F) was calculated as Dose/AUC_(inf). Volume of distribution(V_(z)/F) was determined by taking the ratio between CL/F and λ_(z)(elimination rate constant). Mean residence time (MRT) was estimatedfrom the ratio between AUMC_(inf) and AUC_(inf).

Safety Analyses: Results of physical examinations, vital signs, clinicallaboratory data were summarized in tabular form and presented by patientnumber. Adverse events also were tabulated in a similar fashion.

10. Results

Patient and Study Accountability: Six narcoleptic patients were enrolledand all six completed the study in its entirety.

Protocol Compliance: There were no inclusion criteria violations. Allpatients admitted into the study met the study entrance requirements andcompleted the screening phase at least one day before the treatmentphase.

All six patients took non-study medications in addition tomethylphenidate and GHB doses because none of their concomitantmedications (Synthroid, Premarin, Lovastatin, Fluvastatin, furosemide,potassium, hydrochlorothiazide, Iansoprazole, and verapamil) were on theexclusion list (which included hypnotics, sedatives, antidepressants,antihistamines, clonidine, and anticonvulsants). Adverse experienceprobes, vital sign measurements, and essentially all pharmacokineticblood samples were performed at protocol specified times; the fewdeviations in blood sampling times should not have any impact on theoutcome of the study since actual blood sampling times were used in thepharmacokinetic analysis.

The diagnosis of narcolepsy for at least one year in each patient wasverified by a nocturnal polysomnogram (NSG) and a Multiple Sleep LatencyTest (MSLT) conducted at a qualified laboratory. Five patients have beenmaintained on GHB nightly for over 10 years and one patient has beenreceiving GHB nightly for two years. One patient (Subject 101) also hadmultiple sclerosis; however, the attending physician, judged that itwould not interfere with the objective of this study. A few of thescreening clinical laboratory results marginally fell outside thereference range but none was considered by the attending physician to beclinically significant.

Exposure to Study Drug: All patients ingested the two GHB doses asscheduled (immediately prior to bedtime). The GHB doses per kg bodyweight ranged from 26.4 to 52.4 mg/kg.

Plasma GHB Concentration Profiles: It was noted that, in certain cases,(Patients #103, and #106), plasma GHB concentrations did not declinefrom the first C_(max) to zero concentration at h 4. Upon achievement ofthe second C_(max) the semi-logarithmic plots of concentration versustime data in Patients #102, #103, and #105 exhibited a convex declineprofile. Such a decline pattern suggested non-linear pharmacokinetics.The highest plasma GHB concentration observed in the study was 125.0μg/mL which occurred in Subject 101 after the second 3 g GHB dose.

Pharmacokinetic Parameter Estimates: The mean (±SD) showed that maximumGHB concentrations (C_(max)) were 62.8±27.4 μg/mL and 91.2±25.6 μg/mLfor the first and second GHB doses, respectively. The corresponding meanobserved times to maximum concentrations were 40±6 and 36±7 min afterthe first and second GHB doses, respectively. The mean AUC_(inf) was17732±4603 μg/mL·h. The mean CL/F was 4.2±mL/min/kg and the mean V_(z)/Fwas 307±96 mL/kg. The mean MRT_(inf) was 249±56 min. The mean GHBT_(1/2), estimated by linear regression of log [C] vs. time data of theterminal phase of the second GHB dose was 53±19 min.

Adverse Experiences: No adverse experiences were reported in the study.

Follow-up Safety Assessments: Inspection of screen and follow-upphysical examination results per individual patient did not identify anychanges attributable to GHB.

11. Discussion

To the inventors' knowledge, the level of GHB in human systemiccirculation has not been reported in the literature. Hence, baseline (0h) plasma samples were analyzed for GHB concentrations. The GC-MSDmethod used in the present study had a limit of quantification (LOQ) of7.02 μg/mL and analysis of the baseline plasma samples showed theendogenous levels of GHB are below this sensitivity limit. This findingwas confirmed by adding known amounts of GHB (5, 10, and 25 μg per mL ofplasma) to blank human plasma samples and subjected these samples toGC-MSD analysis. This method of standard addition allowed an estimationof the endogenous GHB level in human plasma which was found to averageabout 2.02 μg/mL, (i.e. approximately 2/7 of the Limit Of Quantitation(LOQ) for a validated assay. Hence, the endogenous GHB level was notsubtracted from exogenous GHB concentrations prior to pharmacokineticanalysis.

Values of mean t_(max) (˜40 min after dosing) and t_(1/2) (˜35 min)suggest that the GHB solution administered to narcoleptic patients inthis study was readily absorbed and rapidly eliminated. In 3 out of 6patients the drug was essentially gone from the systemic circulation byh 4 after the first GHB dose whereas in the remaining three patientsresidual GHB levels of ˜15 μg/mL was still detected at h 4.

The convex nature of the decline of plasma GHB concentrations in threepatients after achievement of the second cmax indicated that eliminationof GHB from the systemic circulation in these three patients is capacitylimited. Nevertheless, it should be noted that plasma GHB concentrationswere no longer detectable by h 6 after the second GHB dose (10 h afterthe first GHB dose). The mean apparent oral clearance found in thisstudy was 4.2±1.0 mL/min/kg and appeared to be comparable to theapparent oral clearance of 5.3±2.2 mL/min/kg reported in the literaturefor a group of alcohol dependent patients who were administered a doseof 50 mg/kg (Ferrara, 1992). While it appeared that the GHB dose(ranging from 26.4 to 52.4 mg/kg with a mean of 36.5 mg/kg) in thepresent study was lower than the comparison GHB dose (50 mg/kg)administered to the alcohol dependent patients (Ferrara, 1992), itshould be noted that each patient in the present study was administeredtwo consecutive GHB doses at four-hour interval and residual GHB levelswere detected in three out of six patients immediately prior to thesecond GHB dose. The GHB pharmacokinetic non-linearity in alcoholdependent patients easily can be observed from the apparent oralclearance which increased to 8.1±4.8 mL/min/kg when the GHB dose isreduced to 25 mg/kg dose (Ferrara, 1992). In the present study, thenon-linearity was less obvious because each narcoleptic patient receivedtwo consecutive fixed 3 g doses regardless of body weight.

The mean elimination half-life of GHB in the six narcoleptic patientswas determined to be 53±19 min, longer than that in alcohol dependentpatients after a 50 mg/kg GHB dose (Ferrara, 1992). The lengthening ofGHB elimination half-life observed in this study partially was caused bythe wider spacing in sampling time points. However, capacity limitedelimination of this drug in some of the narcoleptic patients also couldhave contributed to this prolongation.

GHB appears to have a shortcoming in that its elimination from the bodyis capacity limited in some patients when the drug is administered at afixed regimen of 3 g twice nightly at four-hour interval. However, froma therapeutic perspective, GHB offers an advantage in the treatment ofnarcolepsy because by the time a patient wakes tip in the morning (i.e.8 to 10 h after the first GHB dose), all GHB, including that from thesecond dose, will have been eliminated from the systemic circulation.GHB was also well tolerated by narcoleptic patients in this study. Noadverse experience was reported.

12. Conclusions

The capacity limited elimination kinetics was observed in three out ofsix patients who had been administered two consecutive 3 g oral doses ofGHB, 4 h apart. From a pharmacokinetic perspective, dividing the nightlyGHB dose into two portions and administering the two portions tonarcoleptic patients at a 2.5- to 4-h interval was rational because theelimination half-life of GHB was short (<1 h). The pharmacokineticprofiles of GHB in narcoleptic patients who had been receiving thisagent nightly for years appeared to be comparable to those in alcoholdependent patients (Ferrara, 1992).

Example 4 Sodium Oxybate Formulation Study I. Study Objectives

This example described ways that sodium oxybate may be prepared andtested for stability to determine preferred formulations. Variousformulations of sodium oxybate in water were prepared under differentconditions of mixing and with addition of selected acidulents atmultiple pH levels (Neo-Pharm Laboratories, Blainville, Quebec).Selected formulations were placed on real time and acceleratedstability. Earlier studies have demonstrated that degradation productsare formed in acidic conditions and that antimicrobial effectiveness islimited at high pH. Therefore several acidulents across a range of6.0-9.0 were evaluated.

II. Study Design-Part I

The following experimental work is designed to be performed in twostages. Initial studies were conducted to evaluate the impact ofconditions of formulation, pH and acidulent on the resultant levels ofimpurities, specified and unspecified, and potency of sodium oxybate.Sodium oxybate was prepared (MDS Neo-Pharm Laboratories, Quebec Canada),under different conditions of mixing and with addition of selectedacidulents at multiple pH levels. These formulations of sodium oxybateacidulent were then tested.

A. Preliminary Studies

1. Formulations Description

All formulations were prepared at a concentration of 500 mg/cc of sodiumoxybate in water. Three acidulents (HCl, malic acid, and phosphoricacid), were selected and tested at pH 6.0, 7.5 and 9.0.

2. Method of Formulation

Solutions, were prepared using the described methods:

a. Rapid Mix Method:

Sodium oxybate was dissolved in water and concentrated acidulent wasadded immediately, without temperature control. Temperature of solutionwas monitored and recorded prior to and during addition of acidulent.The time of equilibration to room temperature was also recorded. Afterthe solution reached ambient room temperature, it was filtered through a10 μm filter.

b. Cool Mix Method:

Sodium oxybate was dissolved in water. Acidulent was diluted to 10% andslowly added. The solution was cooled by water with jacket or ice bath.Monitor and record the temperature of the solution was monitored andrecorded during addition of acidulent. The time of equilibrium from roomtemperature was also recorded. The preferred maximum temperature shouldbe maintained at less than 40° C. The solution was filtered through a 10μm filter.

c. Reverse Order of Addition:

Acidulent was added to water and cooled to room temperature. The sodiumoxybate was dissolved in the diluted acidulent solution. The temperatureof solution was monitored and recorded during addition of sodiumoxybate. The solution was filtered through a 10 μm filter.

d. Sodium Oxybate Control:

Sodium oxybate was dissolved in water to a concentration of 500 mg/ccwith no added acidulent. The final pH was recorded and the solution wasfiltered through a 10 μm (micron or micrometer) filter.

3. Solution Data:

Data was recorded for each solution which included: 1) date ofpreparation 2) date of analysis, 3) amount of acidulent required toachieve target pH, 4) length of time for dissolution of sodium oxybate,5) temperature profile of solution over time of solution preparation tobe recorded at 15 minute intervals, 6) final pH of solution.

4. Testing Requirements:

The following methods were used to test the prepared solutions: pH, HPLC(High Pressure Liquid Chromatography) for potency (sodium oxybate), andfor impurities. Time 0 analysis was performed immediately (within 24 h).RRT=(relative retention time).

B. Summary of Part I:

1. Preliminary Evaluation of Sodium Oxybate Formulations

Tables 13, 14 and 15 provide test results for the three methods ofpreparation of sodium oxybate formulations.

Formulation Study/PR98068 Results of Formulation Study—Time ZeroDeterminations of Sodium Oxybate, GBL and Unspecified Impurities

TABLE 13 Preparation Method A Addition of Concentrated Acidulent* SodiumImpurities Impurities (Amount of Acidulent in 1000 ml) Oxybate SpecifiedUnspecified Date of Preparation/Date of Assay Target pH mg/cc % % GBL %[Specification] [Target ± 0.5] Final pH [95-105%] [≦0.5%] [≦0.1% Total]HCl (Apr. 23, 1998) pH 9.0 9.0 509 mg/cc 0.009% RRT 4.88 = 0.01% (10drops over 2 minutes)  101% (2.5 ml/4 minutes) pH 7.5 7.5 507 mg/cc 0.01% RRT 4.89 = 0.02%  101% (45 ml/34 minutes) pH 6.0 6.0 504 mg/cc0.033% RRT 4.89 = 0.33%  101% Malic Acid (Apr. 24, 1998) pH 9.0 9.1 498mg/cc 0.009% RRT 4.89 = 0.01% (0.12 gm) 99.6% (1.6 gm) pH 7.5 7.6 506mg/cc 0.009% RRT 4.89 = 0.01%  101% (25 gm) pH 6.0 6.2 493 mg/cc 0.011%RRT 4.89 = 0.01% 98.6% H₃PO₄ (Apr. 24, 1998) pH 9.0 9.0 493 mg/cc 0.009%RRT 4.89 = 0.01% (2 drops) 98.6% (1.0 ml) pH 7.5 7.5 493 mg/cc 0.009%RRT 4.89 = 0.02% 98.6% (17.3 ml) pH 6.0 6.1 497 mg/cc 0.063% RRT 4.89 =0.02% 99.4% Sodium Oxybate Control n.a. 9.8 500 mg/cc 0.009% RRT 4.89 =0.04% No Acidulent  100% *Method A = Mix with Concentrated Acidulent andTemperature Monitoring

TABLE 14 Preparation Method B Addition of Diluted Acidulent* SodiumImpurities Impurities (Amount of Acidulent in 1000 ml) Oxybate SpecifiedUnspecified Date of Preparation/Date of Assay Target pH mg/ml % % GBL %[Specification] [Target ± 0.5] Final pH [95-105%] [≦0.5%] [≦0.1% Total]HCl (25%) (Apr. 28, 1998) pH 9.0 9.1 500 mg/cc 0.009% RRT 4.88 = 0.01%(20 drops)  100% (8.0 ml) pH 7.5 7.6 499 mg/cc 0.009% RRT 4.88 = 0.01%99.8% (175 ml) pH 6.0 6.0 502 mg/cc 0.016% RRT 4.88 = 0.02%  101% H₃PO₄(25%) (Apr. 29, 1998) pH 9.0 8.9 499 mg/cc 0.007% RRT 4.92 = 0.02% (0.3ml) 99.8% (4.0 ml) pH 7.5 7.5 497 mg/cc 0.008% RRT 4.89 = 0.02% 99.4%(120 ml) pH 6.0 6.0 499 mg/cc 0.019% RRT 4.89 = 0.01% 99.8% Malic Acid(500 mg/cc) (Apr. 30, 1998) pH 9.0 9.0 495 mg/cc 0.008% RRT 4.92 = 0.02%(0.115 gm/0.23 ml)  99% (1.75 gm/3.5 ml) pH 7.5 7.4 488 mg/cc 0.009% RRT4.92 = 0.01% 97.5% (35 gm/70 ml) pH 6.0 6.0 487 mg/cc 0.013% RRT 4.92 =0.01% 97.0% *Acidulent added slowly at the rate of 2-3 drops/second

TABLE 15 Preparation Method C Reverse Order of Addition* SodiumImpurities Impurities (Amount of Acidulent in 1000 ml) Oxybate SpecifiedUnspecified Date of Preparation/Date of Assay Target pH mg/ml % % GBL %[Specification] [Target ± 0.5] Final pH [95-105%] [≦0.5%] [≦0.1% Total]HCl (May 1, 1998) pH 9.0 9.0 497 mg/cc 0.006% RRT 4.92 = 0.03% (20drops) 99.4% (2.4 ml) pH 7.5 7.6 504 mg/cc 0.004% RRT 4.92 = 0.04%  101%(45 ml) pH 6.0 6.0 493 mg/cc 0.044% RRT 4.92 = 0.04% 98.6% H₃PO₄ (May 4,1998) pH 9.0 8.9 496 mg/cc 0.005% RRT 4.91 = 0.03% (0.08 ml) 99.2% (1.0ml) pH 7.5 7.6 496 mg/cc 0.004% RRT 4.91 = 0.04% 99.2% (30 ml) pH 6.06.1 489 mg/cc 0.023% RRT 4.91 = 0.04% 97.8% Malic Acid (May 5, 1998) pH9.0 9.0 495 mg/cc 0.006% RRT 4.93 = 0.02% (0.12 gm)  99% (1.6 gm) pH 7.57.6 497 mg/cc 0.004% RRT 4.93 = 0.04% 99.4% (35 gm) pH 6.0 6.2 495 mg/cc0.044% RRT 4.93 = 0.04%  99% *Acidulent added to water first, GHB addedsecond.

Review of the data indicated that the optimum method for preparation ofsodium oxybate with minimal impurity levels is Method B: Controlledmixing with diluted acidulent. Method 2b resulted in formulations withlowest levels of GBL.

2. Conclusions.

Additional evaluations were carried out on selected formulations: 1)sodium oxybate with HCl as acidulent, at pH 7.5, and 2) sodium oxybatewith malic acid as acidulent, pH 6.0, 7.5, and 9.0.

III. Study Design-Part II

Microbial Challenge and Stability Tested to determine the most preferredembodiments, the number of formulations was limited to three based onthe data prepared from the above experiments.

A. Kinetic Stability Study with Selected Formulations

Samples of formulations are stored in tightly closed containers. StorageConditions were 25° C., 40° C., and 60° C. Time points in brackets weretested at the inventor's discretion. The samples were tested accordingto the following schedule: at 25° C. storage temperature, the assaypoints will be 0, 14, 28, 45, 60 days and 120 days; at 40° C. storagetemperature, the assay points will be 0, 7, 14, 28, 45, 60 days; at 60°C. storage temperature, the assay points will be at 0, 3, 7, 14, 28, 45days, and, 60 days.

The testing requirements included pH, HPLC for sodium oxybate (duplicateinjections of single sample preparation), and impurities, specified andunspecified.

B. Preservative Effectiveness Testing of Selected Formulations

Microbial challenge testing of formulations was preformed according toUSP XXIII, <51>, Eighth Supplement. Solutions are determined to “Pass orFail” based upon the USP criteria for preservative effectiveness whichstates: For Bacteria, “Not less than 1 log reduction from the initialmicrobial count at 14 days and no increase from the 14 days count at 28days;” and for yeast and molds, “No increase from the initial calculatedcount at 14 and 28 days.” Solutions which met these criteria weredesignated as “Pass” and those that did not meet these criteria weredesignated as “Fail”.

C. Summary Stability Results:

1. Formulations Prepared with Malic Acid as Acidulents:

-   -   a. Malic Acid, pH 6.0 formulation (25°), GBL and impurity A        levels were very low an Day 0, however, by Day 45 GBL levels had        reached 2.8%. Impurity A increased from 0.01 to 1.0%, and pH        increased from 6.0 to 6.3 by day 45. This formulation stored at        40° C. and 60° C. showed GBL levels up to 5.4%, impurity A        levels increased to 2.3%, and pH increased to 6.3 by Day 14.    -   b. Malic Acid, pH 7.5 formulation (25° C.), GBL levels were        0.009% on Day 0, and increased to 0.17%0/by day 45. Impurity A        increased from 0.01% to 0.1% and pH increased from 7.5 to 7.9.        Malic acid, pH 7.5 GBL levels are reached (40° C.) and 60° C. a        maximum of 0.22%. Impurity A levels reached 0.1% and pH        increased to 8.0. Under accelerated conditions, all parameters        reached an apparent maximum by Day 7 and did not increase        significantly thereafter.    -   c. Malic Acid, pH 9.0 formulation (25° C.) GBL levels measure        0.008% on Day 0, and increased slightly to 0.013% on Day 45.        Impurity A did not increase nor did pH increase. Under        accelerated conditions, GBL increased from 0.008% to a maximum        of 0.018% by Day 14. Impurity A increased slightly from 0.10 to        0.014% by Day 14.

2. Formulations Prepared with HCl as Acidulents.

HCl, pH 6.0 formulation (250) GBL levels measured 2.8% by Day 30, andimpurity A 0.004%. and pH 6.0. Accelerated storage conditions (40° C.)GBL levels were measured at 6.6%, and impurity A measured 3.1% by Day30.

HCl, pH 7.5 formulation (25%) GBL levels measured 0.041%0/on Day 0,Impurity A measured 0.02%, and by Day 18 GBL measured to 0.12% andimpurity A to 0.07%. Under accelerated conditions (40° C. and 60° C.),GBL increased to a maximum of 0.21%, impurity A increased from 0.02% to0.1%, and pH increased from 7.5 to 8.0. As with Malic Acid at pH 7.5,the measured parameters reached maximum by Day 7 and did not increasesignificantly thereafter.

HCl, Ph 9.0 formulation (25° C.) GBL levels reached 0.022% by Day 18.Impurity A stayed constant at 0.01% for 18 days. Under acceleratedconditions (40° C.) GBL levels were equivalent to 25° C. storage(0.21%). Impurity A showed no increase over 25° C. conditions.

3. Conclusions.

Formulations selected for microbial challenge testing were thefollowing: HCl, Ph 7.5, and malic acid, Ph 7.5. The rationale for thisdecision was twofold. First, the formulations were selected based onminimal formation of GBL and impurity A. Second, the formulations wereselected to maintain a Ph in the neutral range.

Example 5 Further Evaluation of Sodium Oxybate Formulations

Purpose:

To prepare, test and evaluate multiple formulations of Sodium Oxybateand two formulations using alternative salts of gamma-hydroxybultyrate.

Scope:

Various formulations of Sodium Oxybate in water were prepared withaddition of selected acidulents at multiple Ph levels. Solutions wereprepared and tested at Neo-Pharm Laboratories, Blainville. Quebec. Allformulations successfully prepared were placed on limited stability.Earlier studies have demonstrated that degradation products are formedin acidic conditions and that antimicrobial effectiveness is limited athigh Ph. Conditions of varying Ph and concentrations of sodium oxybatepreviously not evaluated were prepared and tested.

Procedures:

Solutions were prepared as summarized and microbial challenge testingcarried out as follows:

B. Evaluation of Sodium Oxybate Formulations

Purpose:

To prepare, test and evaluate multiple formulations of Sodium Oxybateand two formulations using alternative salts of gamma-hydroxybutyrate.

Scope:

Various formulations of Sodium Oxybate in water were prepared withaddition of selected acidulents at multiple Ph levels. Selectedformulations were studied for limited stability. Earlier studiesdemonstrated that degradation products are formed in acidic conditionsand that antimicrobial effectiveness is limited at high Ph. Conditionsof varying Ph and concentrations of sodium oxybate previously notevaluated were prepared and tested.

Responsibility:

It was the responsibility of Neo-Pharm Laboratories to prepare selectedformulations and perform testing per this protocol. Orphan Medical, NewMedicine Development and Quality Assurance were responsible forreviewing raw data at the defined decision point, defining whichformulations will be included in stability testing. Orphan Medical wasalso responsible for reviewing final results (raw data) and the finalreport.

Procedure:

The following formulations were prepared by scientists at Neo-Pharmfollowing the steps listed below and dispensed into containers (amberPET 240 ml bottle, OMI CS-460) and closures (Clic-Loc III, 24-400, OMICS-470) to a volume of 200 ml each bottle. The bottles were tested by28-day microbial challenge and by limited stability testing at 25° C.including appearance, Ph, potency, and impurity profile on day 1 (day ofpreparation) and day 28.

B. Formulations Prepared and Evaluated Using Sodium Oxybate:

TABLE 16 Formulations Prepared and Evaluated Using Sodium OxybateFormulation Sodium Oxybate ID No. Concentration Acidulent Final Ph 1 500mg/cc Malic Acid 7.5 2 250 mg/cc Malic Acid 7.5 3 350 mg/cc Malic Acid7.5 4 450 mg/cc Malic Acid 7.5 5 550 mg/cc Malic Acid 7.5 6 650 mg/ccMalic Acid 7.5 7 500 mg/cc Citric Acid 7.5 8 500 mg/cc Malic Acid 5.0

-   -   1. Preparation: Method for preparation of various formulations:        -   As previously determined in PR98068, the method of choice            for preparation of liquid formulations of sodium oxybate was            the following:        -   a. For a one liter quantity of product, add the sodium            oxybate in 500 ml of purified and stir until dissolved.            Prepare a 100% solution of the acid (Malic or Citric) and            add slowly to the solution of sodium oxybate. The solution            should be monitored for Ph and temperature and both            variables recorded at reasonable intervals (every 10 or 15            minutes). When the target Ph is attained, the solution will            be Q. S. to 1 liter and Ph rechecked and recorded.        -   b. The final solutions will be filtered through 10 μm            filters and 200 Ml dispensed into 5 amber PET bottles with            closures (Orovide by Orphan Medical, Inc.). Two bottles will            be used for microbial challenge studies and the remaining            three bottles will be placed on limited stability.    -   2. Testing: Formulations were tested by two methods of        evaluation:        -   a. Limited stability evaluation:            -   (1) Storage Conditions: 25° C.            -   (2) Pull Points: Day 0 (day of preparation), and day 28            -   (3) Testing Requirements:

Test Method Appearance Visual Potency HPLC Neopharm 764 Impurities HPLCNeopharm 793DT Ph USP <791>

-   -   -   b. Microbial challenge:            -   (1) Storage Conditions: Microbial challenge studies of                above formulations were set up with 5 microorganisms and                stored for 28 days at 20-25° C., per USP <51> Eighth                Supplement.            -   (2) Microorganisms: After a sufficient quantity of each                formulation is prepared, aliquots were inoculated with 5                microorganisms at a concentration of at least 10⁵                microorganisms/cc:                -   (a) Escherichia coli, ATCC 8739                -   (b) Pseudomonas aeruainosa, ATCC 9027                -   ©Staphylococcus aureus, ATCC 6538                -   (d) Aspergillus niger, ATCC 18404                -   (e) Candida albicans, ATCC 10231            -   (3) Time Points: A determination of the viable cell                concentration in each inoculated container was performed                after 0, 1, 3, 7, 14, 21 and 28 days.

B. Formulations To Be Prepared From Alternative Salts ofGamma-Hvdroxvbutvrate: This work may be staged to take place at a latertime than the work described above.

TABLE 17 Formulation Detail Formulation Concentration ID No. Salt of GHBof Salt of GHB Acidultent Final pH 9 Calcium salt 500 mg/cc Malic Acid7.5 (Or maximum (If compatible) possible*)

-   -   1. Solubility determination: Little information is available        about the solubility of this alternative salt of        gamma-hydroxybutyrate and a determination of solubility was done        in advance of efforts to prepare formulations for evaluation by        stability and microbial challenge. Maximum solubility is        evaluated for pH unadjusted solutions and within the pH range        desired for this formulation (pH 6.0-8.0). If solubility is        limited, the formulation will be changed to accommodate the        solubility limitations. The preferred acidulent for this work is        Malic acid. If acid is not compatible with the salt, then an        alternative acid can be selected.    -   2. Preparation: Method for preparation of alternative salt        formulations:        -   a. The previously described method (Part A) is used for            preparation of formulations of calcium gamma-hydroxybutyrate            at the concentrations and specified pH determined by            solubility experiments.        -   b. The final solutions were filtered through 10 μm filters            and dispensed into 5 amber PET bottles with closures            (provided by Orphan Medical, Inc.). Two bottles are used for            microbial challenge studies and two bottles are placed on            limited stability. The remaining bottles are retained for            any additional studies at a future time.    -   3. Testing: Formulations are tested as described above.

C. Reporting of Results: The results will be reported for the Stabilityand Microbial Challenge results in standard format as defined by thedescribed Orphan Medical Development. Copies of HPLC chromatograms andany raw data from these studies will be provided with results.

D. Acceptance Criteria: Specific acceptance criteria for this study canbe described analogous to those for sodium oxybate.

Results: Summarized as follows in Tables 18, 19 and 20 for variousstudies.

TABLE 18 Result Summary Results of Protocol 98126 Microbial ChallengeStudy Lot Number MCH1064-33 GHB, pH 7.50, 500 mg/cc Malic Acid 0 Day 1Day 7 Day 14 Day 21 Day 28 E. coli 490,000 5,500 <100 <10 <10 <10 P.aeruginosa 141,000 21,600 <100 <10 <10 <10 S. aureus 1,035,000 405,00079,500 8,300 1,645 375 C. albicans 835,000 147,000 <100 <10 <10 <10 A.niger 370,000 285,000 120,500 246,500 148,500 183,000 Lot NumberMCH1064-35 GHB, pH 7.50, 250 mg/cc Malic Acid 0 Day 1 Day 7 Day 14 Day21 Day 28 E. coli 705,000 229,500 <100 <10 <10 <10 P. aeruginosa 224,5005,200 <100 <10 <10 <10 S. aureus 1,135,000 390,000 262,500 31,500 4,250155 C. albicans 705,000 435,000 52,000 850 <10 <10 A. niger 510,000515,000 155,500 176,000 147,500 184,000 Lot Number MCH1064-37 GHB, pH7.50, 300 mg/cc Malic Acid 0 Day 1 Day 7 Day 14 Day 21 Day 28 E. coli365,000 310,000 13,400 <10 <10 <10 P. aeruginosa 205,000 15,600 50 <10<10 <10 S. aureus 1,170,500 605,000 67,500 <60 60 <10 C. albicans870,000 355,000 8,300 <10 <10 <10 A. niger 540,000 525,000 172,000155,500 155,500 163,500 Lot Number MCH1064-43 GHB, pH 7.50, 550 mg/ccMalic Acid 0 Day 1 Day 7 Day 14 Day 21 Day 28 E. coli 425,000 63,500 700<10 <10 <10 P. aeruginosa 171,500 211,550 250 <10 <10 <10 S. aureus1,020,000 520,000 41,500 1,050 180 10 C. albicans 880,000 157,500 800<10 <10 <10 A. niger 545,000 505,000 131,000 156,500 205,000 187,500 LotNumber MCH1064-45 GHB, pH 7.50, 550 mg/cc Malic Acid 0 Day 1 Day 7 Day14 Day 21 Day 28 E. coli 660,000 58,500 450 <10 <10 <10 P. aeruginosa896,000 14,450 900 <10 <10 <10 S. aureus 860,000 132,000 19,750 935 11045 C. albicans 1,125,000 166,000 <100 <10 <10 <10 A. niger 530,000530,000 105,500 153,000 157,500 177,000 Lot Number MCH1064-47 GHB, pH7.50, 650 mg/cc Malic Acid 0 Day 1 Day 7 Day 14 Day 21 Day 28 E. coli630,000 119,000 1,350 <10 <10 <10 P. aeruginosa 183,500 5,900 50 <10 <10<10 S. aureus 890,000 650,000 76,000 14,550 510 1,150 C. albicans675,000 145,500 <100 <10 <10 <10 A. niger 535,000 385,000 103,000162,000 187,000 173,000 Lot Number MCH1064-85 Ca-Oxybate, pH 7.50, 500mg/cc Malic Acid 0 Day 1 Day 7 Day 14 Day 21 Day 28 E. coli 425,000121,000 1,650 <10 <10 <10 P. aeruginosa 420,000 22,000 300 <10 <10 <10S. aureus 265,000 2,000 <100 <10 <10 <10 C. albicans 565,000 440,00029,500 <1000 <10 <10 A. niger 1,310,000 965,000 370,000 640,000 690,000675,000 Lot Number MCH1064-49 GHB, pH 7.50, 500 mg/cc Malic Acid 0 Day 1Day 7 Day 14 Day 21 Day 28 E. coli 615,000 6,500 <100 <10 <10 <10 P.aeruginosa 69,500 14,600 <100 <10 <10 <10 S. aureus 650,000 305,0001,700 <10 <10 <10 C. albicans 720,000 107,000 <100 <10 <10 <10 A. niger375,000 380,000 99,500 178,500 212,500 165,500

TABLE 19 Result Summary Data from Dec. 30, 1997 GHB (pH 7.5) (n = 3) 750mg/cc Inoculu 0 Day 1 Day 3 Day 7 Day 14 Day 21 Day 28 E. coli 470,000160,000 64,500 4,300 420 <10 <10 <10 P. aeruginosa 437,500 152,000 3,50010 <10 <10 <10 <10 S. aureus 447,500 330,000 24,500 42,000 8,050 1,93515 10 C. albicans 375,000 234,500 28,000 1,950 <10 <10 10 <10 A. niger475,500 395,000 395,000 229,000 101,500 161,500 101,000 202,000 750mg/cc + 0.2% MP/PP pH 7.50 E. coli 470,000 127,000 <1,000 <10 <10 <10<10 <10 P. aeruginosa 437,500 61,000 <1,000 <10 <10 <10 <10 <10 S.aureus 447,500 350,000 3,000 4,050 <10 <10 <10 <10 C. albicans 375,000103,500 <1,000 <10 <10 <10 <10 <10 A. niger 457,500 315,000 415,00035,500 79,500 38,500 87,500 6,400 750 mg/cc + 0.1% MP/PP, pH 7.5 E. coli470,000 157,000 7,000 <10 <10 <10 <10 <10 P. aeruginosa 437,500 90,000<1,000 <10 <10 <10 <10 <10 S. aureus 447,500 239,000 5,500 16,950 600<10 <10 <10 C. albicans 375,000 169,000 <1,000 <100 <10 <10 <10 <10 A.niger 457,500 335,000 425,000 34,500 168,500 90,500 95,500 99,000 750mg/cc + 0.2% Potassium sorbate, pH 7.5 E. coli 470,000 180,000 735,0006,200 475 <10 <10 <10 P. aeruginosa 437,500 152,000 1,000 <10 <10 <10<10 <10 S. aureus 447,500 264,000 27,500 49,800 14,550 2,370 <10 <10 C.albicans 375,000 300,000 41,500 3,800 <10 <10 <10 <100 A. niger 457,500325,000 360,000 25,000 202,000 500,000 345,000 425,000 GHB (pH 6.0) 500mg/cc Inoculu 0 Day 1 Day 3 Day 7 Day 14 Day 21 Day 28 Results E. coli470,000 221,000 40,000 100 <10 <10 <10 <10 P. aeruginosa 437,500 172,0003,000 <10 <10 <10 <10 <10 S. aureus 447,500 320,000 <1,000 30 <10 <10<10 <10 C. albicans 375,000 310,000 14,000 100 <10 <10 <10 <10 A. niger475,500 270,000 355,000 84,000 120,000 48,500 41,000 8,600 PASS 500mg/cc + 0.2% MP/PP pH 6.0 E. coli 470,000 163,000 <1,000 <10 <10 <10 <10<10 P. aeruginosa 437,500 60,000 <1,000 <10 <10 <10 <10 <10 S. aureus447,500 243,000 <1,000 <10 <10 <10 <10 <10 C. albicans 375,000 150,500<1,000 <100 <10 <10 <10 <10 A. niger 475,500 400,000 38,000 <10 <10 <10<10 <10 PASS 500 mg/cc + 0.1% MP/PP, pH 6.0 E. coli 470,000 206,000<1,000 <10 <10 <10 <10 <10 P. aeruginosa 437,500 118,000 <1,000 <10 <10<10 <10 <10 S. aureus 447,500 330,000 <1,000 <10 <10 <10 <10 <10 C.albicans 375,000 221,000 <1,000 <100 <10 <10 <10 <10 A. niger 475,500355,000 93,500 59,000 8,700 315 35 >10 PASS 500 mg/cc + 0.2% Potassiumsorbate, pH 6.0 E. coli 470,000 222,000 46,500 150 <10 <10 <10 <10 P.aeruginosa 437,500 136,000 <1,000 <10 <10 <10 <10 <10 S. aureus 447,500410,000 <1,000 130 <10 <10 <10 <10 C. albicans 375,000 395,000 28,500<100 <10 <10 <10 <10 A. niger 475,500 405,000 270,000 63,000 51,00049,500 39,000 11,150 PASS

TABLE 20 Result Summary Data from Study Dated Dec. 30, 1997 GHB (pH 6.0)500 mg/cc Inoculum 0 Day 1 Day 3 Day 7 Day 14 Day 21 Day 28 E. coli470,000 221,000 40,000 100 <10 <10 <10 <10 P. aeruginosa 437,500 172,0003,000 <10 <10 <10 <10 <10 S. aureus 447,500 320,000 <1,000 30 <10 <10<10 <10 C. albicans 375,000 310,000 14,000 100 <10 <10 <10 <10 A. niger475,500 270,000 355,000 84,000 120,000 48,500 41,000 8,600 Data FromStudy Begun Mar. 12, 1998 GHB (pH 6.0) 500 mg/cc Inoculum 0 Day 1 Day 3Day 7 Day 14 Day 21 Day 28 E. coli 500,000 370,000 Nd Nd <100 <10 <10<10 P. aeruginosa 350,000 198,500 Nd Nd <100 <10 <10 <10 S. aureus280,000 480,000 Nd Nd <100 <10 <10 <10 C. albicans 450,000 340,000 Nd Nd<100 <10 <10 <10 A. niger 450,000 445,000 Nd Nd 9,050 20,500 9,450 1,120GHB (pH 6.0) 500 mg/cc Inoculum 0 Day 1 Day 3 Day 7 Day 14 Day 21 Day 28E. coli 500,000 199,000 Nd Nd <100 <10 <10 <10 P. aeruginosa 350,000192,500 Nd Nd <100 <10 <10 <10 S. aureus 280,000 300,000 Nd Nd <100 <10<10 <10 C. albicans 450,000 370,000 Nd nd <100 <10 <10 <10 A. niger450,000 445,000 Nd Nd 10,100 22,750 3,800 4,050 Data From Study BegunMar. 12, 1998 GHB (pH 9.0) 500 mg/cc Inoculum 0 Day 1 Day 3 Day 7 Day 14Day 21 Day 28 E. coli 500,000 320,000 Nd Nd <100 <10 <10 <10 P.aeruginosa 350,000 12,000 Nd Nd <100 <10 <10 <10 S. aureus 280,000495,000 Nd Nd <100 <10 <10 <10 C. albicans 450,000 380,000 Nd Nd <100<10 <10 <10 A. niger 450,000 355,000 Nd Nd 12,550 157,500 365,000365,000 GHB (pH 6.0 + Excipients) 500 mg/cc Inoculum 0 Day 1 Day 3 Day 7Day 14 Day 21 Day 28 E. coli 500,000 96,000 Nd Nd <100 <10 <10 <10 P.aeruginosa 350,000 26,000 Nd Nd <100 <10 <10 <10 S. aureus 280,000155,000 Nd Nd <100 <10 <10 <10 C. albicans 450,000 205,000 Nd Nd <100<10 <10 <10 A. niger 450,000 131,500 Nd Nd 6,250 1,825 870 370 GHB (pH6.0 + Excipients) 500 mg/cc Inoculum 0 Day 1 Day 3 Day 7 Day 14 Day 21Day 28 E. coli 500,000 93,000 Nd Nd <100 <10 <10 <10 P. aeruginosa350,000 30,500 Nd Nd <100 <10 <10 <10 S. aureus 280,000 185,000 Nd Nd<100 <10 <10 <10 C. albicans 450,000 135,000 Nd Nd <100 <10 <10 <10 A.niger 450,000 121,500 Nd Nd 5,400 1,785 795 505

TABLE 21 Result Summary GHB (pH 7.50) HCl Jul. 2, 1998 Start Date 500mg/cc Initial Cone 0 Day 1 Day 3 Day 7 Day 14 Day 21 Day 28 E. coli97000 82000 19200 Nd 1000 <10 <10 <10 P. aeruginosa 48500 29500 520 Nd<10 <10 <10 <10 S. aureus 54500 58000 42350 Nd 4950 245 <10 <10 C.albicans 58500 38500 1060 Nd <100 <10 <10 <10 A. niger 77500 48000 21450Nd 46000 46000 38000 54000 GHB (pH 7.50) Malic Acid 500 mg/cc InitialCone 0 Day 1 Day 3 Day 7 Day 14 Day 21 Day 28 E. coli 97000 83000 44450Nd 3050 70 <10 <10 P. aeruginoas 48500 15650 545 Nd <10 <10 <10 <10 S.aureus 54500 59500 48400 Nd 17400 6500 820 505 C. albicans 58500 440006200 Nd 500 <10 <10 <10 A. niger 77500 35500 24100 Nd 28000 49000 4450044000 GHB (pH 7.50) HCl Jul. 2, 1998 Start Date 500 mg/cc Initial Co 0Day 1 Day 3 Day 7 Day 14 Day 21 Day 28 E. coli 9.70E+04 8.20E+041.92E+04 nd 1.00E+03 <10 <10 <10 P. aeruginosa 4.85E+04 2.95E+045.20E+02 nd <10 <10 <10 <10 S. aureus 5.45E+04 5.80E+04 4.24E+04 nd4.95E+03 2.45E+02 <10 <10 C. albicans 5.85E+04 3.85E+04 1.06E+03 nd <100<10 <10 <10 A. niger 7.75E+04 4.80E+04 2.15E+04 nd 4.60E+04 4.60E+043.80E+04 5.40E+04 GHB (pH 7.50) Malic Acid 500 mg/cc Initial Co 0 Day 1Day 3 Day 7 Day 14 Day 21 Day 28 E. coli 9.70E+04 8.30E+04 4.45E+04 nd3.05E+03 7.00E+01 <10 <10 P. aeruginosa 4.85E+04 1.57E+04 5.45E+02 nd<10 <10 <10 <10 S. aureus 5.45E+04 5.95E+04 4.84E+04 nd 1.74E+046.50E+03 8.20E+02 5.05E+02 C. albicans 5.85E+04 4.40E+04 6.20E+03 nd5.00E+02 <10 <10 <10 A. niger 7.75E+04 3.55E+04 2.41E+04 nd 2.80E+044.90E+04 4.45E+04 4.40E+04 For Category IC Products: Bacteria: Not lessthan 1 log reduction from the initial count at 14 days, and no increasefrom the 14 days count at 28 days. Yeast and Molds: No increase from theinitial calculated count at 14 and 28 days.

TABLE 22 pH Variable Result Summary GHB, pH 7.5 GHB, pH 6.0 750 mg/cc500 mg/cc Dec. 30, 1997 Inoculum 0 Day 14 Day 28 Dec. 30, 1997 Inoculum0 Day 14 Day 28 E. coli 470,000 160,000 <10 <10 E. coli 470,000 221,000<10 <10 P. aeruginosa 437,500 152,000 <10 <10 P. aeruginosa 437,500172,000 <10 <10 S. aureus 447,500 330,000 1,935 10 S. aureus 447,500320,000 <10 <10 C. albicans 375,000 234,500 <10 <10 C. albicans 375,000310,000 <10 <10 A. niger 475,500 395,000 161,500 202,000 A. niger475,500 270,000 48,500 8,600 GHB, pH 7.5 GHB, pH 6.0 750 mg/cc + 500mg/cc + 0.2% MP/PP 0.2% MP/PP Dec. 30, 1997 Dec. 30, 1997 E. coli470,000 127,000 <10 <10 E. coli 470,000 163,000 <10 <10 P. aeruginosa437,500 61,000 <10 <10 P. aeruginosa 437,500 60,000 <10 <10 S. aureus447,500 350,000 <10 <10 S. aureus 447,500 243,000 <10 <10 C. albicans375,000 103,500 <10 <10 C. albicans 375,000 150,500 <10 <10 A. niger457,500 315,000 38,500 6,400 A. niger 475,500 400,000 <10 <10 GHB, pH7.5 GHB, pH 6.0 750 mg/cc + 500 mg/cc + 0.1% MP/PP 0.1% MP/PP Dec. 30,1997 E. coli 470,000 157,000 <10 <10 E. coli 470,000 200,000 <10 <10 P.aeruginosa 437,500 90,000 <10 <10 P. aeruginosa 437,500 118,000 <10 <10S. aureus 447,500 239,000 <10 <10 S. aureus 447,500 330,000 <10 <10 C.albicans 375,000 169,000 <10 <10 C. albicans 375,000 221,000 <10 <10 A.niger 457,500 335,000 90,500 99,000 A. niger 475,500 355,000 315 <10GHB, pH 7.5 750 mg/cc + 0.2% GHB, pH 6.0 Potassium 500 mg/cc sorbatexxxxxx Mar. 12, 1998 Inoculum 0 Day 14 Day 28 E. coli E. coli P.aeruginosa P. aeruginosa S. aureus S. aureus C. albicans C. albicans A.niger A. niger GHB, pH 6.0 500 mg/cc + GHB, pH 6.0 0.2% Potassium 500mg/cc sorbate Dec. 30, 1997 Mar. 12, 1998 Inoculum 0 Day 14 Day 28 E.coli 470,000 222,000 <10 <10 E. coli 500,000 199,000 <10 <10 P.aeruginosa 437,500 136,000 <10 <10 P. aeruginosa 350,000 192,500 <10 <10S. aureus 447,500 410,000 <10 <10 S. aureus 280,000 300,000 <10 <10 C.albicans 375,000 395,000 <10 <10 C. albicans 450,000 370,000 <10 <10 A.niger 475,500 405,000 49,500 11,150 A. niger 450,000 445,000 22,7504,050 GHB, pH 6.0 GHB, pH 6.0 500 mg/cc + 500 mg/cc + ExcipientsExcipients Mar. 12, 1998 Inoculum 0 Day 14 Day 28 Mar. 12, 1998 Inoculum0 Day 14 Day 28 E. coli 500,000 93,000 <10 <10 E. coli 500,000 96,000<10 <10 P. aeruginosa 350,000 30,500 <10 <10 P. aeruginosa 350,00026,000 <10 <10 S. aureus 280,000 185,000 <10 <10 S. aureus 280,000155,000 <10 <10 C. albicans 450,000 135,000 <10 <10 C. albicans 450,000205,000 <10 <10 A. niger 450,000 121,500 1,785 505 A. niger 450,000131,500 1,825 370 GHB, pH 9.0 GHB, pH 7.50 500 mg/cc 500 mg/cc HCl Mar.12, 1998 Inoculum 0 Day 14 Day 28 Jul. 2, 1998 Inoculum 0 Day 14 Day 28E. coli 500,000 320,000 <10 <10 E. coli 97000 82000 <10 <10 P.aeruginosa 350,000 12,000 <10 <10 P. aeruginosa 48500 29500 <10 <10 S.aureus 280,000 530,000 <10 <10 S. aureus 54500 58000 245 <10 C. albicans450,000 510,000 <10 <10 C. albicans 58500 38500 <10 <10 A. niger 450,00345,000 158,500 110,500 A. niger 77500 48000 46000 54,000 GHB, pH 9.0GHB, pH 7.5 500 mg/cc 500 mg/cc, Malic Mar. 12, 1998 Inoculum 0 Day 14Day 28 Acid Jul. 2, 1998 Inoculum 0 Day 14 Day 28 E. coli 500,000305,000 <10 <10 E. coli 97000 83000 70 <10 P. aeruginosa 350,000 20,000<10 <10 P. aeruginosa 48500 15650 <10 <10 S. aureus 280,000 495,000 <10<10 S. aureus 54500 59500 6500 505 C. albicans 450,000 380,000 <10 <10C. albicans 58500 44000 <10 <10 A. niger 450,000 355,000 157,500 365,000A. niger 77500 35500 49000 44,000

Short term stability testing was carried out as described in Appendix Aand results are summarized in—Results of Limited StabilityTesting—XYREM® oral solution—are shown as follows:

TABLE 23-A ORPHAN MEDICAL INC. 13911, Ridgedale Drive Minnetonka, (MN)55305 DATE: 26 Jan. 1999 USA NO: 333198 CERTIFICATION OF ANALYSISOXYBATE SODIUM, LIQUID FORM. (28 DAYS CHALLENGE TEST) LOT: MCH1064-3PROTOCOL 98126 CODE: ORPHAN MEDICAL REQUISITION: 1741 TEST SPECIFICATIONRESULT PROCEDURE Description Clear to slightly Conforms ORGANOLEPTICopalescent solution. Potency Report 512 mg/ml (102%) NPLC-793 Impuritiestotal ≦2.0% 0.068% NPLC-793D Impurities specified Gamma-ButyrolactoneRRT 1.45: 0.021% NPLC-793D GBL-RRT 1.6 (RRT = 1.6): ≦0.5% RRT 4.17:0.02% Impurity A (RRT 4.3): ≦0.5% Impurities unspecified Ind. imp. ≦0.1%RRT 1.28: 0.02% NPLC-793D RRT 3.79: 0.007% PH Report 7.6 USP <791>Challenge Test Conforms to USP Conforms USP 23 <51> S.8 (0, 1, 7, 14,21, 28 days) COMMENTS: Initial test Formulation 1: 500 mg/cc; Malicacid; pH 7.5 THIS CERTIFICATE CORRECTS AND REPLACES CERTIFICATE 328841

TABLE 23-B ORPHAN MEDICAL INC. 13911, Ridgedale Drive Minnetonka, (MN)55305 DATE: 21 Jan. 1999 USA NO: 331347 CERTIFICATION OF ANALYSISOXYBATE SODIUM, LIQUID FORMULATION LOT: MCH1064-3 PROTOCOL 98126 CODE:ORPHAN MEDICAL REQUISITION: 1741 TEST SPECIFICATION RESULT PROCEDUREDescription Clear to slightly Conforms ORGANOLEPTIC opalescent solution.Potency Report 510 mg/ml (102%) NPLC-793-D Impurities total ≦2.0% 0.36%NPLC-793-D Impurities specified Gamma-Butyrolactone RRT 1.46: 0.23%NPLC-793-D (RRT = 1.6): ≦0.5% RRT 4.31: 0.1% Impurity A (RRT 4.3): ≦0.5%Impurities unspecified Ind. imp. ≦0.1% *A NPLC-793D PH Report 7.9 USP<791> COMMENTS: 28 days (25° C., 60% RH) Formulation 1: 500 mg/cc; Malicacid; pH 7.5 *A: RRT 1.30: 0.02% RRT 3.93: 0.008%

TABLE 23-C ORPHAN MEDICAL INC. 13911, Ridgedale Drive Minnetonka, (MN)55305 DATE: 26 Jan. 1999 USA NO: 333197 CERTIFICATION OF ANALYSISOXYBATE SODIUM, LIQUID FORMULATION (28 DAYS CHALLENGE TEST) LOT:MCH1064-3 PROTOCOL 98126 CODE: ORPHAN MEDICAL REQUISITION: 1741 TESTSPECIFICATION RESULT PROCEDURE Description Clear to slightly ConformsORGANOLEPTIC opalescent solution. Potency Report 258 mg/ml (102%)NPLC-793-D Impurities total ≦2.0% 0.045% NPLC-793D Impurities specifiedGamma-Butyrolactone RRT 1.45: 0.016% NPLC-793D GBL-RRT 1.6 (RRT = 1.6):≦0.5% RRT 4.17: 0.02% Impurity A (RRT 4.3): ≦0.5% Impurities unspecifiedInd. imp. ≦0.1% RRT 3.79: 0.009% NPLC-793D PH Report 7.6 USP <791>Challenge test Conforms to USP Conforms USP 23 <51> S.8 (0, 1, 7, 14,21, 28 days) COMMENTS: Initial test Formulation 2: 250 mg/cc; Malicacid; pH 7.5 THIS CERTIFICATE CORRECTS AND REPLACES CERTIFICATE 328845

TABLE 23-D ORPHAN MEDICAL INC. 13911, Ridgedale Drive Minnetonka, (MN)55305 DATE: 21 Jan. 1999 USA NO: 331346 CERTIFICATION OF ANALYSISOXYBATE SODIUM, LIQUID FORMULATION (28 DAY CHALLENGE TEST) LOT:MCH1064-3 PROTOCOL 98126 CODE: ORPHAN MEDICAL REQUISITION: 1741 TESTSPECIFICATION RESULT PROCEDURE Description Clear to slightly ConformsORGANOLEPTIC opalescent solution. Potency Report 256 mg/ml (102%)NPLC-793-D Impurities total ≦2.0% 0.18% NPLC-793D Impurities specifiedGamma-Butyrolactone RRT 1.46: 0.13% NPLC-793D (RRT = 1.6): ≦0.5% RRT4.31: 0.03% Impurity A (RRT 4.3): ≦0.5% Impurities unspecified Ind. imp.≦0.1% *A NPLC-793D PH Report 7.9 USP <791> COMMENTS: 28 days (25° C.,60% RH) Formulation 2: 290 mg/cc; Malic acid; pH 7.5 *A: RRT 1.29:0.007% RRT 3.93: 0.008%

TABLE 23-E ORPHAN MEDICAL INC. 13911, Ridgedale Drive Minnetonka, (MN)55305 DATE: 26 Jan. 1999 USA NO: 333196 CERTIFICATION OF ANALYSISOXYBATE SODIUM, LIQUID FORM. (28 DAYS CHALLENGE TEST) LOT: MCH1064-3PROTOCOL 98126 CODE: ORPHAN MEDICAL REQUISITION: 1741 TEST SPECIFICATIONRESULT PROCEDURE Description Clear to slightly Conforms ORGANOLEPTICopalescent solution. Potency Report 360 mg/ml (103%) NPLC-793 Impuritiestotal ≦2.0% 0.050% NPLC-793D Impurities specified Gamma-ButyrolactoneRRT 1.45: 0.017% NPLC-793D GBL-RRT 1.6 (RRT = 1.6): ≦0.5% RRT 4.17:0.02% Impurity A (RRT 4.3): ≦0.5% Impurities unspecified Ind. imp. ≦0.1%RRT 1.28: 0.006% NPLC-793D RRT 3.79: 0.007% PH Report 7.7 USP <791>Challenge test Conforms to USP Conforms USP 23 <51> S.8 (0, 1, 7, 14,21, 28 days) COMMENTS: Initial test Formulation 3: 350 mg/cc; Malicacid; pH 7.5 THIS CERTIFICATE CORRECTS AND REPLACES CERTIFICATE 328847

TABLE 23-F ORPHAN MEDICAL INC. 13911, Ridgedale Drive Minnetonka, (MN)55305 DATE: 21 Jan. 1999 USA NO: 331345 CERTIFICATION OF ANALYSISOXYBATE SODIUM, LIQUID FORMULATION (28 DAYS CHALLENGE TEST) LOT:MCH1064-3 PROTOCOL 98126 CODE: ORPHAN MEDICAL REQUISITION: 1741 TESTSPECIFICATION RESULT PROCEDURE Description Clear to slightly ConformsORGANOLEPTIC opalescent solution. Potency Report 363 mg/ml (104%)NPLC-793-D Impurities total ≦2.0% 0.21% NPLC-793D Impurities Gamma- RRT1.46: 0.14% NPLC-793D specified Butyrolactone RRT 4.31: 0.5% (RRT =1.6): ≦0.5% Impurity A (RRT 4.3): ≦0.5% Impurities Ind. imp. ≦0.1% *ANPLC-793D unspecified PH Report 8.0 USP <791> COMMENTS: 28 DAYS (25° C.,60% RH) Formulation 3: 350 mg/cc; Malic acid; pH 7.5 *A: RRT 1.29:0.009% RRT 3.93: 0.008%

TABLE 23-G ORPHAN MEDICAL INC. 13911, Ridgedale Drive Minnetonka, (MN)55305 DATE: 26 Jan. 1999 USA NO: 333195 CERTIFICATION OF ANALYSISOXYBATE SODIUM, LIQUID FORM. (28 DAYS CHALLENGE TEST) LOT: MCH1064-4PROTOCOL 98126 CODE: 1741 ORPHAN MEDICAL REQUISITION: 1741 TESTSPECIFICATION RESULT PROCEDURE Description Clear to slightly ConformsORGANOLEPTIC opalescent solution. Potency Report 461 mg/ml (102%)NPLC-793 Impurities total ≦2.0% 0.065% NPLC-793D Impurities Gamma- RRT1.45: 0.018% NPLC-793D specified Butyrolactone RRT 4.17: 0.02% GBL-RRT1.6 (RRT = 1.6): ≦0.5% Impurity A (RRT 4.3): ≦0.5% Impurities Ind. imp.≦0.1% RRT 1.28: 0.02% NPLC-793D unspecified RRT 3.79: 0.007% PH Report7.5 USP <791> Challenge test Conforms to USP Conforms USP 23 <51> S.8(0, 1, 7, 14, 21, 28 days) COMMENTS: Initial test Formulation 4: 450mg/cc; Malic acid; pH 7.5 THIS CERTIFICATE CORRECTS AND REPLACESCERTIFICATE 328875

TABLE 23-H ORPHAN MEDICAL INC. 13911, Ridgedale Drive Minnetonka, (MN)55305 DATE: 21 Jan. 1999 USA NO: 331343 CERTIFICATE OF ANALYSIS OXYBATESODIUM, LIQUID FORMULATION LOT: MCH1064-4 PROTOCOL 98126 CODE: ORPHANMEDICAL REQUISITION: 1741 TEST SPECIFICATION RESULT PROCEDUREDescription Clear to slightly Conforms ORGANOLEPTIC opalescent solution.Potency Report 454 mg/ml (101%) NPLC-793-D Impurities total ≦2.0% 0.40%NPLC-793D Impurities Gamma- RRT 1.46: 0.26% NPLC-793D specifiedButyrolactone RRT 4.31: 0.1% (RRT = 1.6): ≦0.5% Impurity A (RRT 4.3):≦0.5% Impurities Ind. imp. ≦0.1% *A NPLC-793D unspecified PH Report 7.8USP <791> COMMENTS: 28 DAYS (25° C., 60% RH) Formulation 4: 450 mg/cc;Malic acid; pH 7.5 *A: RRT 1.30: 0.03% RRT 3.93: 0.008%

TABLE 23-I ORPHAN MEDICAL INC. 13911, Ridgedale Drive Minnetonka, (MN)55305 DATE: 26 Jan. 1999 USA NO: 333194 CERTIFICATE OF ANALYSIS OXYBATESODIUM, LIQUID FORM. (28 DAYS CHALLENGE TEST) LOT: MCH1064-4 PROTOCOL98126 CODE: ORPHAN MEDICAL REQUISITION: 1741 TEST SPECIFICATION RESULTPROCEDURE Description Clear to slightly Conforms ORGANOLEPTIC opalescentsolution. Potency Report 563 mg/ml (102%) NPLC-793 Impurities total≦2.0% 0.077% NPLC-793D Impurities Gamma- RRT 1.45: 0.020% NPLC-793Dspecified Butyrolactone RRT 4.17: 0.02% GBL-RRT 1.6 (RRT = 1.6): ≦0.5%Impurity A (RRT 4.3): ≦0.5% Impurities Ind. imp. ≦0.1% RRT 1.29: 0.03%NPLC-793D unspecified RRT 3.79: 0.007% PH Report 7.6 USP <791> Challengetest Conforms to USP Conforms USP 23 <51> S.8 (0, 1, 7, 14, 21, 28 days)COMMENTS: Initial test Formulation 5: 550 mg/cc; Malic acid; pH 7.5 THISCERTIFICATE CORRECTS AND REPLACES CERTIFICATE 328883

TABLE 23-J ORPHAN MEDICAL INC. 13911, Ridgedale Drive Minnetonka, (MN)55305 DATE: 21 Jan. 1999 USA NO.: 331341 CERTIFICATE OF ANALYSIS OXYBATESODIUM, LIQUID FORMULATION LOT: MCH1064-4 PROTOCOL 98126 CODE: ORPHANMEDICAL REQUISITION: 1741 TEST SPECIFICATION RESULT PROCEDUREDescription Clear to slightly Conforms ORGANOLEPTIC opalescent solution.Potency Report 561 mg/ml (102%) NPLC-793-D Impurities total ≦2.0% 0.56%NPLC-793D Impurities Gamma- RRT 1.46: 0.31% NPLC-793D specifiedButyrolactone RRT 4.31: 0.2% (RRT = 1.6): ≦0.5% Impurity A (RRT 4.3):≦0.5% Impurities Ind. imp. ≦0.1% *A NPLC-793D unspecified PH Report 7.9USP <791> COMMENTS: 28 DAYS (25° C., 60% RH) Formulation 5: 550 mg/cc;Malic acid; pH 7.5 *A: RRT 1.30: 0.04% RRT 3.93: 0.007%

TABLE 23-K ORPHAN MEDICAL INC. 13911, Ridgedale Drive Minnetonka, (MN)55305 DATE: 26 Jan. 1999 USA NO: 333193 CERTIFICATE OF ANALYSIS OXYBATESODIUM, LIQUID FORM. (28 DAYS CHALLENGE TEST) LOT: MCH1064-4 PROTOCOL98126 CODE: ORPHAN MEDICAL REQUISITION: 1741 TEST SPECIFICATION RESULTPROCEDURE Description Clear to slightly Conforms ORGANOLEPTIC opalescentsolution. Potency Report 666 mg/ml (102%) NPLC-793 Impurities total≦2.0% 0.10% NPLC-793D Impurities Gamma- RRT 1.45: 0.025% NPLC-793Dspecified Butyrolactone RRT 4.17: 0.02% GBL-RRT 1.6 (RRT = 1.6): ≦0.5%Impurity A (RRT 4.3): ≦0.5% Impurities Ind. imp. ≦0.1% RRT 1.28: 0.05%NPLC-793D unspecified RRT 3.78: 0.007% PH Report 7.6 USP <791> Challengetest Conforms to USP Conforms USP 23 <51> S.8 (0, 1, 7, 14, 21, 28 days)COMMENTS: Initial test Formulation 6: 650 mg/cc; Malic acid; pH 7.5 THISCERTIFICATE CORRECTS AND REPLACES CERTIFICATE 328885

TABLE 23-L ORPHAN MEDICAL INC. 13911, Ridgedale Drive Minnetonka, (MN)55305 DATE: 21 Jan. 1999 USA NO: 331336 CERTIFICATE OF ANALYSIS OXYBATESODIUM, LIQUID FORMULATION LOT: MCH1064-4 PROTOCOL 98126 CODE: ORPHANMEDICAL REQUISITION: 1741 TEST SPECIFICATION RESULT PROCEDUREDescription Clear to slightly Conforms ORGANOLEPTIC opalescent solution.Potency Report 660 mg/ml (102%) NPLC-793 Impurities total ≦2.0% 0.81%NPLC-793D Impurities Gamma- RRT 1.46: 0.43% NPLC-793D specifiedButyrolactone RRT 4.31: 0.3% (RRT = 1.6): ≦0.5% Impurity A (RRT 4.3):≦0.5% Impurities Ind. imp. ≦0.1% *A NPLC-793D unspecified PH Report 7.8USP <791> COMMENTS: 28 DAYS (25° C., 60% RH) Formulation 6: 650 mg/cc;Malic acid; pH 7.5 *A: RRT 1.30: 0.07% RRT 3.93: 0.007%

TABLE 23-M ORPHAN MEDICAL INC. 13911, Ridgedale Drive Minnetonka, (MN)55305 DATE: 26 Jan. 1999 USA NO: 333192 CERTIFICATE OF ANALYSIS OXYBATESODIUM, LIQUID FORM. (28 DAYS CHALLENGE TEST) LOT: MCH1064-4 PROTOCOL98126 CODE: ORPHAN MEDICAL REQUISITION: 1741 TEST SPECIFICATION RESULTPROCEDURE Description Clear to slightly Conforms ORGANOLEPTIC opalescentsolution. Potency Report 518 mg/ml (102%) NPLC-793 Impurities total≦2.0% 0.065% NPLC-793D Impurities Gamma- RRT 1.45: 0.018% NPLC-793Dspecified Butyrolactone RRT 4.17: 0.02% GBL-RRT 1.6 (RRT = 1.6): ≦0.5%Impurity A (RRT 4.3): ≦0.5% Impurities Ind. imp. ≦0.1% RRT 3.79: 0.007%NPLC-793D unspecified RRT 5.99: 0.02% PH Report 7.5 USP <791> Challengetest Conforms to USP Conforms USP 23 <51> S.8 (0, 1, 7, 14, 21, 28 days)COMMENTS: Initial test Formulation 7: 500 mg/cc; Malic acid; pH 7.5 THISCERTIFICATE CORRECTS AND REPLACES CERTIFICATE 329033

TABLE 23-N ORPHAN MEDICAL INC. DATE: 21/01/1999 13911, Ridgedale DriveNO: 331335 Minnetonka, (MN) 55305 USA CERTIFICATE OF ANALYSIS OXYBATESODIUM, LIQUID LOT: MCH1064-4 FORMULATION CODE: PROTOCOL 98126REQUISITION: 1741 ORPHAN MEDICAL TEST SPECIFICATION RESULT PROCEDUREDescription Clear to slightly Conforms ORGANOLEPTIC opalescent solution.Potency Report 515 mg/ml (101%) NPLC-793-D Impurities total ≦2.0% 0.38%NPLC-793D Impurities Gamma- RRT 1.46: 0.27% NPLC-793D specifiedButyrolactone RRT 4.31: 0.1% (RRT = 1.6): ≦0.5% Impurity A (RRT 4.3):≦0.5% Impurities Ind. imp. ≦0.1% 3.93: 0.007% NPLC-793D unspecified PHReport 7.9 USP <791> COMMENTS: 28 DAYS (25° C., 60% RH) Formulation 7:500 mg/cc; Malic acid; pH 7.5

TABLE 23-O ORPHAN MEDICAL INC. DATE: 09/02/1999 13911, Ridgedale DriveNO: 330721 Minnetonka, (MN) 55305 USA CERTIFICATE OF ANALYSIS OXYBATECALCIUM, LIQUID LOT: MCH1064-85 FORM. CODE: (28 DAYS CHALLENGE TEST)REQUISITION: 1741 PROTOCOL 98126 ORPHAN MEDICAL TEST SPECIFICATIONRESULT PROCEDURE Description Clear to slightly Conforms ORGANOLEPTICopalescent solution. Challenge Test Conforms to USP Conforms USP 23 <51>S.8 (0, 1, 7, 14, 21 and 28 days) Potency Report 501 mg/ml (100%)NPLC-793 Impurities total ≦2.0% 1.2% NPLC-793D Impurities Ind. imp.≦0.1% *A NPLC-793D unspecified Impurities Gamma- RRT 1.46: 0.013%NPLC-793D specified Butyrolactone GBL-RRT 1.6 Report: PH Report 7.3 USP<791> Solubility study Report *B PR 98126 IIA COMMENTS: Initial test 500mg/cc; Malic acid; pH 7.5 *A: RRT 1.31: 0.02% RRT 1.67: 0.008% RRT 1.91:Interference with peak dilution solvent cannot calculate RRT 3.47: 0.1%RRT 3.79: 0.009% RRT 3.84: 0.01% RRT 4.18: 0.06% RRT 5.10: 0.008% RRT5.35: 0.02% RRT 6.74: 0.9% RRT 6.90: 0.08% RRT 7.41: 0.006% *B: Maximumsolubility: 700 mg/ml no pH adjustment.

TABLE 23-P ORPHAN MEDICAL INC. DATE: 26/02/1999 13911, Ridgedale DriveNO: 331307 Minnetonka, (MN) 55305 USA CERTIFICATE OF ANALYSIS OXYBATECALCIUM, LIQUID LOT: MCH1064-85 FORM. CODE: PROTOCOL 98126 REQUISITION:1741 ORPHAN MEDICAL TEST SPECIFICATION RESULT PROCEDURE DescriptionClear to slightly Conforms ORGANOLEPTIC opalescent solution. PotencyReport 508 mg/ml NPLC-793 (102%) Impurities total ≦2.0% 0.70% NPLC-793DImpurities Ind. imp. ≦0.1% *A NPLC-793D unspecified Impurities Gamma-RRT 1.37: NPLC-793D specified Butyrolactone 0.054% Report: PH Report 7.6USP <791> COMMENTS: 28 DAYS (25° C., 60% RH) 500 mg/ml cc; Malid acid;pH 7.5 *A: RRT 1.17: 0.03% RRT 3.47: 0.2% RRT 5.46: 0.01% RRT 6.87: 0.3%RRT 7.04: 0.007% RRT 1.78: Can not calculate because it interfere with adilution solvent peak.

This report summarizes the results of the above described study andprovides a summary of previous development work which evaluatedconditions other than those evaluated in this study. The purposes ofthis information is to define the scope and limitations of theself-preserving properties of Xyrem® oral solution for completion ofpatent application.

II. Summary of Results:

A. Preparation of Various Formulations of Sodium Oxybate andFormulations Using an Alternative Salt of GHB.

-   -   1. Various formulations of sodiwn oxybate were prepared as        directed in the above Protocol. Sodium oxybate. 500 mg/cc with        Malic Acid was not soluble at pH 5.0, and further evaluation of        this solution was discontinued. All other solutions were        successfully prepared as described.    -   2. The preparation of an alternative salt of        gamma-hydroxybutyrare was described as the calcium salt,        prepared at 500 mg/cc (or maximum possible) with Malic Acid at        pH 7.5.        -   a. The calcium salt of gamma-hydroxybutyrate was prepared by            Toronto Research and shipped to NeoPharm for determination            of solubility and evaluation according to the Protocol. The            absolute limit of solubility, without pH adjustment, was            determined to be 700 mg/cc. The pH of this solution was 8.4.            Solutions of lower pH were more difficult to prepare at 500            mg/cc using Malic acid, as acidulant. When pH was adjusted            to 6.0 with Malic acid. the solubility of the calcium            oxybate was limited (longer stirring required to            solubilize). The desired solution of 500 mg/cc, pH 7.5 was            prepared with Malic acid as acidulant without difficulty.            Appearance of the final solution was slightly yellow in            color. Copies of the laboratory record for preparation of            these solutions is available.

B. Microbial Challenge Testing of the Various Formulations Parepared byMDS NeoPharm.

The microbial challenge testing was carried as specified in the Protocoland the following table summarizes the results of microbial challengetesting of various formulations of sodium oxybate and the single calciumoxybate formulation prepared.

TABLE 24 Testing of Sodium and Calcium GHB Salts Microbial Challenge pHof Solution Result Sodium Oxybate Concentration 1. 500 mg/cc 7.5 (Malicacid) Pass 2. 250 mg/cc 7.5 (Malic acid) Pass 3. 350 mg/cc 7.5 (Malicacid) Pass 4. 450 mg/cc 7.5 (Malic acid) Pass 5. 550 mg/cc 7.5 (Malicacid) Pass 6. 650 mg/cc 7.5 (Malic acid) Pass 7. 500 mg/cc 7.5 (Citricacid) Pass Calcium Oxybate Concentration 500 mg/cc 7.5 Pass

C. Short Term Stability Evaluation of Various Formulations of SodiumOxybate and a Formulation of Calcium Oxybate.

Solutions were tested on day zero (preparation doy) nnd day 28 accordingto the described Protocol. The results of the stability evaluation aresummarized in Table 25 below:

TABLE 25 Sodium and Calcium GHB Evaluation Sodium Impurities oxybatePotency Impurities Impurities (Specified- solution mg/cc (%) (Total)(Unspecified) GBL) pH 500 mg/cc 512 mg/cc  0.68% 0.041% 0.027% 7.6 pH7.5 (102%) Malic Acid Day 0 Day 28 510 mg/cc  0.36%  0.33% 0.028% 7.9(103%) 250 mg/cc 258 mg/cc 0.045% 0.009% 0.026% 7.6 pH 7.5 (103%) MalicAcid Day 0 Day 28 256 mg/cc  0.18% 0.015%  0.16% 7.9 (102%) 350 mg/cc360 mg/cc 0.050% 0.013% 0.037% 7.7 pH 7.5 (103%) Malic Acid Day 0 Day 28363 mg/cc  0.21% 0.017%  0.19% 8.0 (104%) 450 mg/cc 461 mg/cc 0.065%0.027% 0.038% 7.5 pH 7.5 (102%) Malic Acid Day 0 Day 28 454 mg/cc  0.40%0.038%  0.36% 7.8 (101%) 550 mg/cc 563 mg/cc 0.077% 0.037% 0.040% 7.6 pH7.5 (102%) Malic Acid Day 0 Day 28 561 mg/cc  0.56% 0.047%  0.51% 7.9(102%) 650 mg/cc 666 mg/cc  0.10% 0.057% 0.045% 7.6 pH 7.5 (102%) MalicAcid Day 0 Day 28 660 mg/cc  0.81% 0.077%  0.73% 7.8 (102%) 500 mg/cc518 mg/cc 0.065% 0.027% 0.038% 7.5 pH 7.5 (104%) Citric Acid Day 0 Day28 515 mg/cc  0.38% 0.007% 0.037% 7.9 (103%) 500 mg/cc 501 mg/cc  ½% >0.1% (See 0.013% 7.3 pH 7.5 (100%) C of A Malic Acid Attached) Day0 Day 28 508 mg/cc  0.70%  >0.1% 0.054% 7.6 (102% (See C of A)

D. Summary of Pertinent Solubility and Microbial Challenge Data areshown in Tables 26 and 27.

TABLE 26 Limits of Solubility pH of Solution Comments Sodium oxybateMaximum Solubility 450 mg/cc pH 4 (HCl) 25° 500 mg/cc pH 5 (HCl) 25° 600mg/cc pH 6 (HCl) 25° 750 mg/cc pH 6.8 (HCl) 25° 1000 mg/cc pH(unadjusted) 65° Soluble, 25° gel Calcium oxybate Maximum Solubility 700mg/cc pH 8.4 (unadjusted) 25° 500 mg/cc pH 6.0 25°

TABLE 27 Microbial Challenge Results Microbial Challenge pH of SolutionResults Sodium oxybate Concentration (Date) 750 mg/cc (Dec. ′97) 7.5(HCl) pass 500 mg/cc (Dec. ′97) 6.0 (HCl) pass 500 mg/cc + Excipients6.0 (Malic Acid) pass (Xylitol) (March ′98) 500 mg/cc (March ′98) 9.0(HCl) pass (Borderline aspergillus) 150 mg/cc (BDL 1995) 5.0 (HCl) fail(aspergillus only) 150 mg/cc (BDL 1995) 7.0 (HCl) fail (aspergillus andstaph) 150 mg/cc (BDL 1995) 3.0 (HCl) fail (aspergillus only) 150 mg/cc(BDL 1995) 10.3 (HCl) fail (aspergillus and staph) 500 mg/cc (May 1998)6.0 (Malic Acid) discontinued 500 mg/cc (May 1998) 7.5 (Malic Acid) pass500 mg/cc (May 1998) 9.0 (Malic Acid) discontinued 500 mg/cc (May 1998)7.5 (HCl) pass 500 mg/cc 7.5 (Malic Acid) pass 250 mg/cc 7.5 (MalicAcid) pass 350 mg/cc 7.5 (Malic Acid) pass 450 mg/cc 7.5 (Malic Acid)pass 550 mg/cc 7.5 (Malic Acid) pass 650 mg/cc 7.5 (Malic Acid) pass 500mg/cc 7.5 (Citric Acid) pass Calcium oxybate Concentration (Date) 500mg/cc 7.5 (Malic Acid) pass

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 5,380,937-   U.S. Pat. No. 4,393,236-   German Patent DD 237,309 Al Brant et al., “Preparation of    storage-stable sodium gamma.-hydroxybutyrate”-   British Pat. No. 922,029-   Aden and Stock, “Increases in brain dopamine,” 1973.-   Arena and Fung, “Absorption of soidum γ-hydroxybutyrate and its    prodrug γ-butyrolactone: relationship between in vitro transport and    in vivo absorption,” J. Pharmaceutical Sciences, 69(3):356-358,    1980.-   Bedard, Montplaisir, Godbout, Lapierre, “Nocturnal    γ-Hydroxybutyrate—Effect on Periodic Leg Movements and Sleep    Organization of Narcoleptic Patients,” 12(1):29-36, 1989.-   Berthier, Bonneau, Desbordes, Chevrel, Oriot, Jaeken, Laborit,    “Possible involvement of a gamma-hydroxybutyric acid receptor in    startle disease,” Acta Paediatr., 83:678-80, 1994.-   Broughton and Mamelak, “The treatment of narcolepsy-cataplexy with    nocturnal gamma-hydroxybutyrate,” Le Journal Canadien Des Sciences    Neurologiques. 6(1):1-6, 1979.-   Ferrara, Zotti, Tedeschi, Frison, Castagna, Gallimberti, Gessa,    “Pharmacokinetics of γ-hydroxybutyric acid in alcohol dependent    patients after single and repeated oral doses,” Br. J Clin.    Pharmacol., 34:231-235, 1992.-   Gallimberti, Canton, Tentile, Ferri, Cibin, Ferrara, Fadda, Gessa,    Gamma-hydroxybutyric acid for treatment of alcohol withdrawal    syndrome,” Clinical Pharmacalagy, 787-789, 1989;-   Gallimberti, Ferri, Ferrara, Fadda, Gessa “Gamma-Hydroxybutyric acid    in the treatment of alcohol dependence: a double-blind study,”    Alcohol Clin. Exp. Res., 16(4):673-676, 1992;-   Gallimberti et al., “Gamma-Hydroxybutyric Acid in the Treatment of    Alcohol Dependence: A Double-Blind Study” Clin. Exp. Res., 16,    673-676, 1992.-   Gessa and Gallimberti, “Gamma-hydroxybutyric acid in the treatment    of alcohol dependent,” Clin. Neuropharm., 15(1,PtA):303A-304A, 1992.-   Gessa, Diana, Fadda, Colombo, “Gamma-hydroxybutyric acid (GHB) for    treatment of ethanol dependence,” Clin. Neuropharm.—Supplement,    1992.-   Gessa et al., Clin. Neuropharm., 15(supp.):303A-304A, 1992-   Gessa et al., Internat. Clin. Psychopharm., 1994-   Grove-White and Kelman, “Critical Flicker Frequency after small    doses of Methohexitone, Diazepam and Sodium 4-Hydroxybutyrate, Brit.    J Anaesth., p. 43, 110, 1971.-   Grove-White and Kelman, “Effect of Methohexitone, Diazepam and    Sodium 4-Hydroxybutyrate on Short-Term Memory,” Brit. J.    Anaesth., p. 43, 113, 1971.-   Hasenbos and Gieien, “Anaesthesia for bullectomy,” Anaesthesia,    40:977-980, 1985.-   Hoes, Vree, Guelen, “Gamma-hydroxybutyric acid (.) as hypnotic,”    L'Encephale. 4(1); 93-99, 1980.-   Laborit, “Gamma-Hydroxybutyrate, Succinic Semi aldehyde and Sleep,”    Laboratojre d' Eutonologie, H{hacek over (o)}pital Boucicaut, Paris    15, France, 1973.-   Ladinsky. Consolo, Zatta, Vezzani. “Mode of Action of    Gamma-Butyrolactone on the Central Cholinergic System,    Naunyn-Schmiedeberg's Arch. Pharmacal., 322:42-48, 1983.-   Ladinsky et al., “Increases in brain acetylcholine,” 1983.-   Lammers et al., “Gammahydroxybutyrate and narcolepsy: a double-blind    placebo-controlled study,” Sleep, 16(3); 216-220, 1993.-   Lapierre, Montplaisir, Lamarre, Bedard, “The Effect of    Gamma-Hydroxybutyrate on Nocturnal and Diurnal Sleep of Normal    Subjects: Further Considerations on REM Sleep-Triggering    Mechanisms,” Sleep. 13(1):24-30, 1990.-   Lapierre et al., “Increases in delta sleep,” 1988.-   Lapierre et al., “Increases in delta sleep,” 1990.-   Lee, C. R. “Evidence for the β-Oxidation of Orally Administered    4-Hydroxybutyrate in Humans” Biochem. Medicine 17, 284-291, 1977.-   Lettieri and Fung, “Improved pharmacological activity via pro-drug    modification: comparative pharmacokinetics of sodium    γ-hydroxybutyrate and γ-butyrolactone,” Research Communications In    Chemical Pathology and Pharmacology. 22(1):107-118. 1978.-   Mamelak, 1977; “Effects Of Gamma Hydroxybutyrate On Sleep,” Biol.    Psychiatry 12, 273-288.-   Mamelak, “Gamma-hydroxybutyrate (GHB): An endogenous regulator of    energy metabolism,” Neuroscience and Biobehav. Reviews, 13: 189-198,    1989.-   Mamelak, “Gammahydroxybutyrate: An Endogenous Regulator of Energy    Metabolism,” Neuro. & Biobehav. Rev., 13 187-198, 1989.-   Mamelak, 1979;-   Mamelak, Escriu, Stokan “The effects of gamma-hydroxybutyrate on    sleep,” Biol. Psychiatry, 12(2):273-288. 1977.-   Mamelak, Escriu, Stokan, “Sleep-Inducing Effects of    Gammahydroxybutyrate,” The Lance, p. 328-329. 1973.-   Nema, et al., “Excipients and their use in injectable products,”    PDA J. Plarm. Sci. Technol., 51(4):166-171, 1997.-   Palatini, Tedeschi, Frison, Padrini, Zordan, Orlando, Gallimberti,    Gessa, Ferrara, “Dose dependent absorption and elimination of    gamma-hydroxybutyric acid in healthy volunteers,” Eur. J Clin.    Pharmacol., 45:353-356, 1993.-   Roth and Giarman, “γ-Butyrolactone and γ-Hydroxybutyric Acid-I,    Distribution and Metabolism,” Biochemical Pharmacology,    15:1333-1348, 1966.-   Scharf, Brown, Woods, Brown, Hirschowitz, “The effects and    effectiveness of gammahydroxybutyrale In patients with    narcolepsy,”, J. Clin. Psychiatry, 46(6)222-225, 1985.-   Scrima, Hartman, Johnson, Miller, “Gamma-Hydroxybutyrate Effects on    Cataplexy and Sleep Attacks in Narcoleptics,” Abstract, p. 134.-   Scrima, Hartman, Johnson, Thomas, Hiller, “Efficacy of    gamma-hydroxybutyrale versus placebo in treating    narcolepsy-cataplexy: Double-blind subjective measured,” Biol.    Psychiatry, 26:331-343, 1989.-   Scrima, Hartman, Johnson, Thomas, Hiller, “The Effects of    γ-Hydroxybutyrate on the Sleep of Narcolepsy Patients: A    Double-Blind Study,” Sleep, 13(6):479-490, 1990.-   Scrima, Hartman, Johnson, Thomas, Miller, “Effects of    Gamma-Hydroxybutyrate (GHB) on Narcolepsy-Cataplexy Symptoms and    MSLT Results in Male and Female Patients,” Abstract, p. 251.-   Scrima, Hoddes, Johnson, Cardin, Thomas, Miller, “Effect of High Alt    itude on a Patient with Obstructive Sleep Apnea,” Abstract, p. 427.-   Scoma, Hoddes, Johnson, Miller, “Effect of Gamma-Hydroxybutyrate on    a Patient with Obstructive Sleep Apnea,” Abstract, p. 137.-   Scrima, et al, “Sleep Res. 16, 134, 1987, Abstract.-   Sériès, Sériès, Cormier, “Effects of Enhancing Slow-Wave Sleep by    Gamma-Hydroxybutyrate on Obstructive Sleep Apnea,” Am. Rev. Respir.    Dis., 1378-1383, 1992.-   Snead and Morley, “Ontogeny of gamma-hydroxybutyric acid. Regional    concentration in developing rat, monkey and human brain,” Brain    Res., 227:579-589, 1981.-   Sneed, Anticonvulsants, alcohol abuse and opiate withdrawal, 1988-   Stock, Magnusson, Anden, “Increase in Brain Dopamine after Axotomy    or Treatment with Gammahydroxybutyric Acid due to Elimination of the    Nerve Impulse Flow,” Naunyn-Schmiedeberg's Arch. Pharmacol., 278,    347-361, 1973.-   Strong, “γ-Hydroxybutyric acid and Intracranial Pressure,” The    Lancet, Vol. 1: No. 8389, 1984.-   van den Bogert, Vree, van der Kleijn, Damsma, “Placentatransfer of    4-Hydroxybutyric Acid in Man.”-   Vickers, “Gammahydroxybutyric Acid,” Int. Anesth. Clinic, 7:75-89,    1969.-   Lee, Biochem. Med., 17:234-291, 1977.-   Yamada, Yamamoto, Fujiki, Hishikawa, Kaneko, “Effect of    Butyrolactone and Gamma-Hydroxybutyrate on the EEG and Sleep Cycle    in Man,” Electroenceph. Clin. Neurophysiol., 22:558-562, 1967.-   Yamada et al., 1967.

1. A pharmaceutical composition, comprising a solution of between about350 and 750 mg/ml of a salt of gamma-hydroxybutyrate, wherein thecomposition has a pH of between about 7.0 to about 9.0, wherein thecomposition is chemically stable and resistant to microbial growth andfurther consists of a preservative selected from the group consisting ofsodium benzoate, methylparaben, propyl gallate BP, sorbic acid,chlorobutanol, dihydroacetic acid, monothioglycerol, potassium benzoate,benzoic acid, benzalkonium chloride, benzethonium chloride, benzylalcohol, butylparaben, cetylpyridinium chloride, ethylenediamine,ethylparaben, glycerin, hypophophorus acid, phenylmercuric nitrate,sassafras oil, sodium propionate, thimerosal, magnesium stearate,magnesium carbonate and potassium sorbate.